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Testing the Nearest Neighbor Model for Canonical RNA Base Pairs: Revision of GU Parameters

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† Department of Chemistry, University of Rochester, Rochester, New York 14627, United States
‡ Department of Chemistry, Allegheny College, Meadville, Pennsylvania 16335, United States
§ Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, United States
# Center for RNA Biology, University of Rochester, Rochester, New York 14627, United States
*Phone: (585) 275-3207. Fax: (585) 276-0205. E-mail: [email protected]
Cite this: Biochemistry 2012, 51, 16, 3508–3522
Publication Date (Web):April 10, 2012
https://doi.org/10.1021/bi3002709
Copyright © 2012 American Chemical Society
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Abstract

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Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5′GG/3′UU motif were added to the single representation in the previous database. This revises the ΔG°37 for the 5′GG/3′UU motif from an unfavorable 0.5 kcal/mol to a favorable −0.2 kcal/mol. Similarly, the ΔG°37 for the 5′UG/3′GU motif changes from 0.3 to −0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°37, ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.

  Funding Statement

This work was supported by NIH Grant GM22939 (D.H.T.)

⊥ Author Present Address

Department of Chemistry, Northwestern University, Evanston, Illinois, 60208, USA.

∥ Author Present Address

Roswell Park Cancer Institute, Buffalo, New York 14263.

The explosion of biological data in the genomics era has filled databanks with large amounts of genetic information. Understanding of these data and making correlations are vital for maximally advancing the fields of biology and medicine. This necessitates accurate methods in bioinformatics and computational chemistry. One important area that bioinformatics and computational chemistry address is finding, predicting, and determining RNA structure from sequence.(1)
RNA participates in a variety of cellular functions involving gene expression and regulation. RNA typically folds in a hierarchical way.(2, 3) Base pairs form to generate motifs such as helixes and loops. Higher order interactions between these features result in three-dimensional structures. On that basis, knowledge of secondary structure is critical for the prediction of tertiary structure. Secondary structure prediction algorithms utilizing experimental thermodynamic data(4-9) have relied on nearest neighbor models.(10-13) Finding regions of genome sequences that code for structured RNA often also relies on nearest neighbor models.(1, 14-16) Because RNA molecules and their reverse complements can fold similarly, the thermodynamics of GU pairs provides information about the reading direction because their complement, CA, forms less stable base pairs.(17)
Prediction of GU pairs is also important because they are the most common non-Watson–Crick pair and have functions in a wide variety of RNAs. For example, GU pairs are found within two helical regions and at the junction of a helix and multibranch loop in eukaryotic 5S rRNA.(18, 19) A GU pair in the third position of the acceptor stem in tRNAAla(20) distorts helix geometry(21) and is important in Escherichia coli for recognition by alanine aminoacyl tRNA synthetase.(22-24) Local helix geometry due to a conserved GU pair may also be important for binding of a yeast intron with hPrp8 or L32 protein.(25, 26) The U-rich tail of guide RNAs bind to a purine-rich region in unedited pre-mRNA to generate recurring 5′AGA/3′UUU motifs that may help RNA editing proteins bind to the major groove.(27, 28) The 5′ leader of HIV-1 can switch between helixes containing GU pairs to promote translation or packaging of its genome.(29)
GU pairs expose the exocyclic amino group of guanine in the minor groove, presenting a unique site for hydrogen bonding to facilitate function and molecular recognition. For example, a GU amino group at the splice site in the Tetrahymena thermophilia group I intron helps bind and align the splice site(30-32) and stabilize the transition state of the splicing reaction.(33) Šponer et al. reported a common tertiary interaction involving a GU pair, where the exocyclic NH2 of the G and the 2′OH of the U form hydrogen bonds, respectively, with the 2′OH and carbonyl oxygen of a cytidine in a GC pair of another helix.(34)
GU pairs can be metal ion binding sites.(35-39) Colmenarejo and Tinoco observed that Co(NH3)63+ preferably binds to 5′GU/3′UG and 5′GG/3′UU over 5′UG/3′GU pairs, whereas Mg(H2O)62+ binds tightest to 5′UG/3′GU.(40) This preference may explain why 5′UG/3′GU is the most prevalent tandem GU motif in rRNA.(41) The propensity for binding metal ions allows design of sequences that bind heavy metals to facilitate solving of X-ray structures.(37, 38)
Prediction of GU pairs often relies on a nearest neighbor model for folding stability. The database of RNA sequences from which GU nearest neighbor parameters were derived(12) is relatively small, however, compared to that for Watson–Crick nearest neighbors.(11) To expand the database, optical melting experiments were carried out on 29 oligoribonucleotide duplexes. Linear regression analysis on the expanded database provides a revised set of individual nearest neighbor (INN) parameters,(42) which are reported herein. The parameters provide stability increments for internal and single terminal GU pairs. Stability increments for additional terminal GU pairs have been reported by Nguyen and Schroeder.(43)

Materials and Methods

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Design of Oligonucleotides

Oligonucleotides were designed to expand the previous database(12) to provide all possible combinations of base pair triplets containing GU pairs flanked by Watson–Crick pairs in different orientations (Table 1) and to have a substantial representation of each nearest neighbor containing a GU pair. An additional six sequences containing the 5′GG/3′UU doublet provided nine new representations for that motif, which had only one representation. Care was taken to select self-complementary sequences that do not favorably form alternative secondary structures, such as hairpins or loops.
Table 1. Occurrence of Each Base Pair Triplet 5′WGY/3′XUZ in the Database of RNA Sequences from Which INN Parameters for GU Pairs Were Derived
WX/YZAUCGGCUAGUUG
AU232416
CG244327
GC823241
UA422222
GU251100
UG455600

Synthesis and Purification of Oligoribonucleotides

Sequences for the following oligoribonucleotide duplexes were purchased from Integrated DNA Technologies (IDT): r(AGGCUU)2, r(AUGCGU)2, r(AGUCGAUU)2, r(CUGGCUAG)2, r(5′CAGAGGAGAC/3′GUCUUUUCUG), r(CAGUCGAUUG)2, r(CCGAAUUUGG)2, r(CGGAAUUUCG)2, r(CGGAUAUUCG)2, r(CGGGCGUUCG)2, r(CUGGAUUCAG)2, r(GAGAGCUUUC)2, r(GAGGAUCUUC)2, r(5′GAGUGGAGAG/3′CUCAUUUCUC), r(GGUUCGGGCC)2, and r(GUGAAUUUAC)2 (the / denotes a nonself-complementary duplex). Purity was checked by NMR except for those forming duplexes with adjacent GU pairs, which were checked by thin layer chromatography. All other sequences were synthesized and purified as previously described.(44) All sequences were desalted with Sep-Pak C18 cartridges (Supporting Information).

UV Melting

RNA duplexes with concentrations from 10–6–10–3 M were melted in 0.5 mM Na2EDTA, 1 M NaCl, and 20 mM sodium cacodylate, pH 7, which maintains a stable pKa over a wide temperature range.(45) Absorbance at 280 nm, typically from 15 to 80 °C, was measured on a Beckman Coulter DU 640 spectrophotometer.

NMR Experiments

Spectra were acquired on a Varian Inova 500 or 600 MHz spectrometer. The buffer for NMR was 80 mM NaCl, 18.8 mM NaH2PO4, 1.16 mM Na2HPO4, 0.02 mM Na2EDTA, pH 6.0, to which 15 μL of D2O was added to provide a lock signal. One-dimensional 1H spectra were acquired with the water 1H signal suppressed with a binomial 1:1 shaped pulse.(46) Two-dimensional 1H–1H NOESY and 1H–1H TOCSY spectra were acquired with the water signal suppressed by a WATERGATE-type pulse sequence with flipback.(47, 48) Two-dimensional 1H–1H NOESY spectra for r(AUGCGU)2 were also measured in D2O.
Spectra were processed with NMRPipe(49) and resonances were assigned with SPARKY.(50) Proton chemical shifts were referenced to a temperature-dependent water chemical shift, δ,(1)where T is temperature in Kelvin.(51) The internal reference standard for water was 2,2-dimethylsilapentate-5-sulfonic acid.

Melting Data Analysis

Melting curves for each duplex were fit to a two-state model with MeltWin 3.5(52) to derive values for ΔH° and ΔS°. The melting temperature, TM, was plotted against ln(CT/a) to provide another measure of ΔH° and ΔS°:(2)Here R is the gas constant (1.987 cal K–1 mol–1), CT is the total concentration of strands, and a is 1 for self-complementary duplexes and 4 for non-self-complementary duplexes. Sequences were added to the database if ΔH° values derived from averaging fits of melting curves agreed within 15% with these derived from eq 2, consistent with the two-state model.

Linear Regression to Fit Nearest Neighbor Parameters

Nearest neighbor thermodynamic parameters were obtained with a regression function reported by Xia et al.(11) Matrix calculations were performed with R(53) and independently verified with Mathematica 8.0(54) and Octave.(55) All three software packages yielded nearly identical results.
Terms representing free energy contributions from non-GU nearest neighbors, that is, helix initiation (ΔG°init), symmetry (ΔG°sym), terminal AU pairs (ΔG°term AU), and Watson–Crick nearest neighbors (ΔG°j (WC NN)),(11) were subtracted from the free energy found from the TM–1 vs ln(CT/a) plots (ΔG°i(duplex)) to provide an experimental free energy attributable to the GU components of each duplex:(3)where i and j are labels for each different duplex and INN parameter, respectively, NN stands for nearest neighbor parameter, and mij is the number of terminal AU pairs. For example,(4)Here, ΔG°37(GU component) contains four 5′GG/3′CU nearest neighbors and two 5′GG/3′UU nearest neighbors. Values for Watson–Crick nearest neighbors from Xia et al.(11) were used because experimental measurements on 22 duplexes not included in the fitting by Xia et al.(11) are predicted within experimental error (Supporting Information). Making the new GU parameters consistent with the Xia et al.(11) parameters provides compatibility with loop parameters derived with Xia et al.(11) nearest neighbor parameters and allows easy adoption by programs using those parameters.
Each experimental duplex ΔG°37 was given an error limit of ±4% to account for systematic errors unless the percent difference between parameters found from TM–1 vs ln(CT/a) and averaged curve fits was greater. For the seven latter cases, this percent difference was doubled to provide an error limit. Error limits for ΔH° were assumed to be 12%.(11) The symmetry contribution, 0.43 kcal/mol in ΔG°37, has no error(56) and was therefore subtracted from ΔG°37 of self-complementary duplexes before calculating the error limit.
The GU component free energies were placed into M × 1 matrix G, where M is the number of duplexes.(5)S is an M × N matrix containing the counts of each nearest neighbor doublet in a duplex, where N is the number of GU nearest neighbor parameters being fit. GNN is an N × 1 matrix that contains the nearest neighbor parameters to be derived from G and S.
The general law of error propagation was used to calculate the variances for each duplex.(57, 58) Multiplication of both sides of eq 5 by an M × M matrix, σ–1, containing the variances in the diagonals yielded error-weighted matrices from which thermodynamic parameters were derived.(6)The values in GNN are thus Sσ–1·Gσ. The variances of each INN parameter are obtained with singular value decomposition (SVD) (ref 11, Supporting Information). Nearest neighbor parameters for ΔH° were found through the same process, and ΔS° parameters were calculated from ΔS° = (ΔH° – ΔG°)/TM.
Nearest neighbor parameters for Watson–Crick pairs were obtained from fitting published data for 112 duplexes, which included the 90 duplexes that Xia et al. previously fit, and 22 additional duplexes (Supporting Information). The symmetry contribution, if present, was subtracted from each thermodynamic parameter derived from the TM–1 vs ln(CT/a) plot. Matrix calculations were carried out as described above to generate ΔG°37 and ΔH° for each nearest neighbor parameter, with all three software packages yielding similar results.
The F-test was used to test the hypothesis that a least-squares model can fit the dependence of Gσ on Sσ and GNN.(59, 60) If the F-value is larger than the critical F-value for N and N − v degrees of freedom at the 5% significance level, where N is the number of duplexes and v the number of nearest neighbor parameters, or if the p-value is less than 0.05, then the hypothesis that there is a dependence of Gσ on GNN may be accepted.(60)
The paired t-test was used to evaluate the significance of the differences between predictions of thermodynamic properties with the updated parameters and those reported by Mathews et al.(12) and the difference between experimental values and predictions by each set of nearest neighbor parameters. The difference between each pair of a set with b values of a variable, X, before and after treatment is defined as μ(XD) = μ(X1) – μ(X2), where X2 represents the response of X1 to treatment.(61) The null hypothesis states that μ(XD) = 0. To test this and the alternative hypothesis that μ(XD) ≠ 0, the mean and standard deviation of the difference between each block of values is found.(7)(8)A t-ratio is defined as(9)
If the t-ratio is greater than t-value for (b – 1) degrees of freedom or less than its negative, then the null hypothesis is rejected at the 0.05 significance level.
For example, in using the paired t-test to evaluate how well experimental ΔG°’s are predicted by nearest neighbor parameters, b is the number of duplexes whose ΔG°’s are being tested and XD is the difference between the predicted and experimental ΔG° for each sequence.
The probability density function (PDF), f(t), of the Student’s t-distribution was used as a measure of how significantly a given INN parameter contributes to the model,(11, 59) with smaller values of f(t) indicating greater contribution,(10)where Γ is the gamma function, r = N – v degrees of freedom and t = ΔG°j(NN)/σj(NN), that is, the quotient of the free energy of the INN parameter over the estimate of its error. Calculations were carried out with R(53) using the anova and t-test functions, and the critical t-value was determined with the qt function in R.

Results

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Table 2 lists results for duplexes in the database used for determination of nearest neighbor parameters for GU pairs. Most of the duplexes are six to eight base pairs in length and have melting temperatures in the 30–70 °C range. For the 29 new duplexes reported here, the average difference between ΔG°37, ΔH°, and ΔS° derived from TM–1 vs ln(CT/a) plots and averaged curve fits are 2%, 7%, and 8%, respectively. Three duplexes, r(AGGCUU)2, r(AUGCGU)2, and r(GUCGUAC/), with TM’s less than 25 °C that were included in the database of Mathews et al.(12) were omitted from the new database. Determination of thermodynamics from optical melting curves is difficult when the TM is less than 25 °C.
Table 2. Thermodynamic Parameters for Duplex Formation in 1 M NaCla
TM–1 vs log CTaverage of curve fitspredicted 
sequenceb–ΔG°37(kcal/mol)–ΔH° (kcal/mol)–ΔS° (eu)TMc(°C)–ΔG°37(kcal/mol)–ΔH° (kcal/mol)–ΔS° (eu)TMc(°C)–ΔG°37(kcal/mol)–ΔH° (kcal/mol)–ΔS° (eu)TMc(°C)ref
Two State Sequences Used in Regression Analysis
CGGCUG5.5543.20121.435.75.5145.40128.635.94.9441.27117.131.7101
CUGCGG4.3141.40119.626.84.5536.00101.427.44.9441.27117.131.7101
GCCGGUp9.1758.20158.157.09.4460.40164.357.08.6655.99152.754.4102
GCGUGC5.1146.18132.433.25.1549.69143.633.74.1051.10151.527.986
GCUGGC6.4759.10169.741.56.5959.10169.341.96.4357.67165.041.4101
GGCGCU8.4256.40154.752.98.4755.40151.353.18.2255.67152.852.1102
GGCGUC4.6738.10107.829.04.9237.30104.430.25.7443.27120.937.6101
GUGCAU5.1047.50136.933.15.1047.00135.033.44.9246.93135.532.043
UCCGCC/6.7157.00162.238.06.6954.30153.437.97.7348.54131.644.784
UCCGGGp7.4447.70129.848.57.3447.10128.248.77.9647.87128.752.5102
UGGCCGp8.5653.00143.354.78.1146.60124.155.17.9249.17133.051.8102
UUGCAG4.2037.20106.525.34.3035.50100.425.73.8240.07116.923.343
CUCGCUC/7.7864.20181.843.18.0070.30200.843.68.1758.88163.446.0103
GCGGGAC/9.0045.20116.855.09.3050.60133.154.79.8562.22168.754.8this work
AGUCGAUU6.0053.30152.638.96.0358.20168.338.94.1447.17138.827.1104
AUGCGCGUp9.3154.90147.058.69.0553.90144.658.88.0855.25152.151.1101
AUGCGUAUp5.2746.80133.934.45.2942.60120.334.84.2242.45123.326.6101
AUGUGCAUp6.1757.10164.239.56.0851.30145.839.95.8763.41185.737.7101
CAGGGCUC/11.1062.80166.661.411.5065.60174.261.011.5271.58193.660.4this work
CCAGUUGG5.7061.10178.637.15.8060.40176.337.46.2065.80192.339.395
CCAUGUGG7.8070.50202.146.57.8071.20204.346.98.5971.47202.950.095
CCUGUAGG6.8171.10207.342.06.8166.10191.142.46.2259.88173.139.7104
CGGAUUCG6.5672.60213.040.86.5970.30205.441.15.9261.87180.338.3104
CGUUGACG6.9373.50214.642.46.9368.40198.042.86.2773.87217.939.7104
CUCGGCUC/8.2273.90211.844.28.3076.80220.944.38.4276.70220.144.9104
CUGGCUAG7.1060.38171.844.47.0662.56179.044.07.3853.03147.147.5this work
GACGCCAG/10.5063.80171.857.511.0074.60205.156.911.3571.19192.959.5this work
GACGCGUU9.5062.20169.957.49.6062.70171.457.69.1276.11215.951.9this work
GAGGUGAG/7.6378.40228.241.47.6376.10220.841.67.1068.65198.439.7105
GAGUGCUC9.4083.00237.451.69.2077.40220.051.89.2177.05218.752.052
GAGUGGAG/9.6682.30234.149.39.5980.40228.249.39.2675.40213.248.8105
GAUGCAUUp6.8262.90180.842.66.8458.70167.243.26.3471.67210.739.9106
GCAGCUGU10.3072.30199.858.310.4072.00198.458.911.5875.29205.563.3this work
GCAGUUGC5.9064.80190.038.16.0069.30203.938.66.5268.78200.740.985
GCAUGUGC8.4072.40206.149.28.5073.00208.049.48.9174.45211.351.185
GCUGGUGC/7.6069.40199.142.07.7071.80206.642.18.4873.24208.845.585
GGAGCUCU10.5066.57180.961.110.6067.60184.061.311.3075.99208.462.0this work
GGAGUUCC6.4373.10214.940.26.4468.40199.640.56.6869.80203.541.5104
GGAUGUCC8.3973.00208.449.08.5978.00223.449.09.0775.47214.151.6104
GGCGGGGC/13.8076.50202.269.514.0077.40204.470.112.5485.75236.060.444
GGCGUGCC9.7273.40206.955.09.7574.20208.055.010.6277.88216.958.0104
GGCUGGCC13.1087.20238.865.913.3090.30248.465.412.9584.45230.466.452
GGUUGACC8.3078.30225.947.68.3077.40222.847.78.0779.37229.946.752
GUAGCUAU7.3050.30138.747.47.4057.90162.846.77.5262.39176.946.5this work
GUCGGGCC/15.0096.00261.366.914.2083.30222.768.613.1175.61201.466.8this work
GUCGUGAC6.0569.10203.338.76.0864.70188.939.06.4469.02201.740.7104
GUCUAGAU7.7070.00201.046.37.7068.70196.846.37.4764.70184.545.9107
UACCGGUG9.7051.70135.463.110.0054.70144.163.69.6258.53157.759.5this work
UAUGCAUGp6.4462.30180.141.06.5553.10150.141.86.1052.83150.739.5106
UCACGUGG8.4046.90124.256.08.6045.00117.158.710.1464.77176.160.1this work
UGACGUCG10.4063.80172.361.610.6067.30182.761.79.5265.83181.456.5this work
UUACGUAG6.2044.60124.040.56.2049.20138.940.15.6853.13152.937.2this work
CAGAGGAGAC/9.4398.95288.646.49.3995.19276.746.610.2396.23277.349.4this work
CAGCGCGUUG12.3177.02208.666.211.5966.84178.267.112.8483.53227.966.1this work
CAGUCGAUUG8.7092.33269.747.58.5484.54245.047.99.2675.49213.652.4this work
CCAGCGUCCU/11.6087.90246.055.911.7090.10252.755.913.1780.81218.264.6108
CCGAAUUUGG6.9976.76225.042.67.0076.35223.542.68.4878.07224.548.4this work
CGGAAUUUCG7.8890.52266.544.77.7784.47247.344.97.7875.51218.345.9this work
CGGAUAUUCG8.7888.20256.248.38.4980.30231.448.58.3578.94227.548.0this work
CGGGCGUUCG11.55101.66290.556.011.65101.34289.256.410.96100.19287.754.3this work
CGGUGCAUCG14.76102.42282.664.114.1894.72259.764.213.2482.27222.668.4this work
CUGGAUUCAG10.1597.81282.751.89.9792.86267.352.09.5882.43234.952.4this work
GAGAGCUUUC8.8286.57250.648.98.7282.81238.948.99.4885.79245.951.5this work
GAGGAUCUUC9.8393.86270.951.39.4083.92240.351.410.2282.33232.355.4this work
GAGUGGAGAG/9.8796.93280.748.19.7993.52270.048.210.2493.27267.649.9this work
GGUUCGGGCC13.59115.71329.359.813.56113.36321.860.212.76105.69299.759.2this work
GUGAAUUUAC4.7862.63186.432.84.6072.40218.632.54.7264.89193.932.6this work
GUGUGCAUAC8.9058.60160.155.09.2066.70185.654.210.1871.65198.257.9this work
GUUAGCUGAC8.6069.60196.750.48.5066.90188.550.59.3477.71220.352.5this work
UCGCCAGAGG/15.3293.46252.069.215.4294.49254.969.216.3592.38245.273.8109
a

Duplexes with a 5′GGUC/3′CUGG motif are not listed because they were not used in fitting nearest neighbor parameters and no new sequences were measured.

b

Listed in order of length of the oligoribonucleotide and then alphabetically for sequences of the same length. All non-self-complementary sequences have a slash and only one sequence shown. GU base pairs are underlined.

c

Calculated at a total strand concentration of 1 × 10–4 M.

With the Exclusion of the 5′GGUC/3′CUGG Motif, the Experimental Results Can Be Fit to a Nearest Neighbor Model

The results in Table 2 were fit to a nearest neighbor model for GU pairs after subtracting contributions from Watson–Crick nearest neighbors (eq 3). This method avoids conflating thermodynamic parameters for Watson–Crick pairs with the idiosyncrasies of GU pairs. Published thermodynamics for duplexes with all Watson–Crick pairs (Supporting Information) were used to test published parameters for Watson–Crick pairs.(11) Fitting the expanded database of 112 duplexes gave INN parameters within error of the values reported by Xia et al.(11) (Table 3). Most free energy parameters did not change by more than 0.05 kcal/mol at 37 °C. Consequently, the GU component values were calculated from eq 3 by subtracting the previously published Watson–Crick thermodynamic values(11) so that the GU parameters are consistent with the widely used Watson–Crick parameters.
Table 3. INN Parameters for Canonical Base Pairs in 1 M NaCl without a Separate Parameter for Terminal GU Pairs
INNINN countsΔG°37(kcal/mol)ΔG°37 error (kcal/mol)ΔH° (kcal/mol)ΔH° error (kcal/mol)ΔS°a(eu)ΔS° error (eu)
Watson–Crick Nearest Neighbor Doublets
5′AA3′ –0.93 (−0.96)0.03 (0.03)–6.82 (−7.09)0.79 (0.77)–19.0 (−19.8)2.5 (2.4)
3′UU5′
5′AU3′ –1.10 (−1.09)0.08 (0.07)–9.38 (−9.11)1.68 (1.56)–26.7 (−25.8)5.2 (4.8)
3′UA5′
5′UA3′ –1.33 (−1.39)0.09 (0.08)–7.69 (−8.50)2.02 (1.86)–20.5 (−22.9)6.3 (5.7)
3′AU5′
5′CU3′ –2.08 (−2.07)0.06 (0.06)–10.48 (−10.90)1.24 (1.15)–27.1 (−28.5)3.8 (3.5)
3′GA5′
5′CA3′ –2.11 (−2.11)0.07 (0.06)–10.44 (−11.03)1.28 (1.18)–26.9 (−28.8)3.9 (3.6)
3′GU5′
5′GU3′ –2.24 (−2.27)0.06 (0.05)–11.40 (−11.98)1.23 (1.12)–29.5 (−31.3)3.9 (3.5)
3′CA5′
5′GA3′ –2.35 (−2.39)0.06 (0.05)–12.44 (−13.21)1.20 (1.05)–32.5 (−34.9)3.7 (3.2)
3′CU5′
5′CG3′ –2.36 (−2.38)0.09 (0.09)–10.64 (−10.88)1.65 (1.54)–26.7 (−27.4)5.0 (4.7)
3′GC5′
5′GG3′ –3.26 (−3.31)0.07 (0.06)–13.39 (−14.18)1.24 (1.07)–32.7 (−35.0)3.8 (3.3)
3′CC5′
5′GC3′ –3.42 (−3.46)0.08 (0.07)–14.88 (−16.04)1.58 (1.33)–36.9 (−40.6)4.9 (4.0)
3′CG5′
GU Nearest Neighbor Doublets
5′GU3′80.720.19–13.834.21–46.913.0
3′UG5′
5′GG3′9–0.250.16–17.823.75–56.711.6
3′UU5′
5′AG3′22–0.350.08–3.961.73–11.65.3
3′UU5′
5′UG3′18–0.390.09–0.961.80–1.85.5
3′AU5′
5′UU3′26–0.510.08–10.381.79–31.85.5
3′AG5′
5′UG3′10–0.570.19–12.644.01–38.912.3
3′GU5′
5′AU3′24–0.900.08–7.391.65–21.05.1
3′UG5′
5′CG3′26–1.250.09–5.561.68–13.95.1
3′GU5′
5′CU3′21–1.770.09–9.441.76–24.75.4
3′GG5′
5′GG3′24–1.800.09–7.031.75–16.85.4
3′CU5′
5′GU3′25–2.150.10–11.091.78–28.85.4
3′CG5′
5′GGUC3′b3–4.120.54–30.808.87–86.023.7
3′CUGG5′
Other Nearest Neighbor Parameters
initiationc4.09 (4.23)0.22 (0.20)3.61 (6.40)4.12 (3.56)–1.5 (6.99)12.7 (10.9)
terminal AU penaltyc0.45 (0.43)0.04 (0.04)3.72 (3.85)0.83 (0.77)10.5 (11.04)2.6 (2.4)
symmetryc0.43000–1.40
a

Values for ΔS° were derived from ΔS° = (ΔH° – ΔG°37)/310.15.

b

Ref 12.

c

Values for initiation, terminal AU, and nearest neighbors with only Watson–Crick pairs are from ref 11 when not in parentheses and derived from an expanded database when in parentheses. Values for nearest neighbors with GU pairs were derived using the Xia et al. parameters(11) for Watson–Crick nearest neighbors.

For fitting GU parameters, duplexes containing the motif, 5′GGUC/3′CUGG, were excluded from the regression due to its poor fit in the nearest neighbor model.(12) For the other 70 duplexes, 12 GU INN parameters were initially derived by linear regression, which included a penalty term for terminal GU pairs to correct for the fact that two duplexes with the same nearest neighbors can have different numbers of GU pairs and therefore different number of hydrogen bonds. A similar term was required for terminal AU pairs.(11) Fitting of additional parameters would not give a unique fit.(42) This 12 parameter fit gave values of −0.02 ± 0.06 kcal/mol and 2.34 ± 1.17 kcal/mol for the terminal GU penalty ΔG°37 and ΔH°, respectively (Supporting Information). The PDF for the terminal GU penalty ΔG°37 and ΔH° were 0.38 and 5.6 × 10–2, respectively, indicating that the term is not statistically significant. Therefore, the data were fit without a terminal GU term. The resulting nearest neighbor parameters are listed in Table 3.
The free energy parameters at 37 °C for 5′UG/3′AU, 5′UU/3′AG, and 5′AU/3′UG are less favorable than previously reported(12) by at least 0.43 kcal/mol. This corresponds to at least a factor of 2 for an equilibrium constant at 37 °C. The ΔG°37 values for each of the tandem GU motifs are more favorable than previously reported. The 5′UG/3′GU nearest neighbor contributes favorably to helix stability by −0.57 kcal/mol, whereas previous data provided an unfavorable increment of 0.30 kcal/mol.(12) Similarly increased stability from 0.47 to −0.25 kcal/mol at 37 °C was found for 5′GG/3′UU, which was previously represented by a single duplex (Table 3).
Estimated errors of the free energy parameters for most nearest neighbor motifs are less than 0.10 kcal/mol (Table 3). The p-value for the F-test is less than 2.2 × 10–16, indicating that there is a linear dependence of the free energy of a duplex on the occurrence of each nearest neighbor parameter in it at the 5% significance level.(60) The PDF values from the Student t-distribution (Table 4) are small for ΔG°37 except for the 5′GG/3′UU motif. The relatively large PDF for the 5′GG/3′UU motif may be attributed to the small magnitude of its free energy and large error compared to those of most of the other INN parameters.
Table 4. Probability Density Function (PDF) of Student’s t-Distribution for ΔG°37 and ΔH° for Each GU INN Motif without a Separate Parameter for Terminal GU Pairsa
motifPDF, ΔG°37PDF, ΔH°motifPDF, ΔG°37PDF, ΔH°
5′GU3′5.8 × 10–42.6 × 10–35′AU3′4.7 × 10–166.1 × 10–5
3′UG5′3′UG5′
5′GG3′1.2 × 10–12.4 × 10–55′CG3′4.9 × 10–202.4 × 10–3
3′UU5′3′GU5′
5′AG3′8.7 × 10–53.1 × 10–25′CU3′1.8 × 10–272.7 × 10–6
3′UU5′3′GG5′
5′UG3′1.0 × 10–43.4 × 10–15′GG3′7.4 × 10–282.8 × 10–4
3′AU5′3′CU5′
5′UU3′5.9 × 10–85.3 × 10–75′GU3′1.6 × 10–291.0 × 10–7
3′AG5′3′CG5′
5′UG3′5.6 × 10–33.7 × 10–3   
3′GU5′
a

Refs 11 and 59. The 5′GGUC/3′CUGG motif is not included.

Table 5 lists results for apparently two-state duplexes that were omitted from the database fitted because their TM’s are less than 25 °C. The predicted thermodynamic parameters for r(AGGCUU)2 and r(AUGCGU)2 do not agree well with those measured. The NMR spectra of r(AGGCUU)2 and r(AUGCGU)2 have strong H2′-H6/8 cross peaks and a sequential H2′, H1′-H6/8 proton walk in 2D 1H–1H NMR (Supporting Information) that indicate the duplexes adopt a largely A-form conformation.(62) For r(AUGCGU)2, however, the presence of broad on-diagonal peaks and exchange cross peaks in the aromatic region of the 2D spectra and of more imino resonances in a 1D spectrum than the number of imino protons in the sequence indicates the presence of alternate conformations. The presence of broad on-diagonal peaks, particularly for A1H8 and H2, in the aromatic region of the 2D spectra for r(AGGCUU)2 also suggests multiple conformations at 0 °C.
Table 5. Thermodynamic Parameters for Formation of Duplexes Not Included in Fitting Nearest Neighbor Parametersa
 TM–1 vs log CTaverage of curve fitspredicted 
sequenceb–ΔG°37(kcal/mol)–ΔH° (kcal/mol)–ΔS° (eu)TMc (°C)–ΔG°37(kcal/mol)–ΔH° (kcal/mol)–ΔS° (eu)TMc (°C)–ΔG°37(kcal/mol)–ΔH°(kcal/mol)–ΔS° (eu)TMc (°C)ref
Sequences with TM < 25 °C
AGGCUU4.0737.10106.624.24.2434.6097.924.72.2430.6391.55.8104
AUGCGUp4.2224.5065.419.54.3724.1063.621.12.3029.7388.55.2101
GUCGUAC/4.20 ± 0.6547.30 ± 9.01139.1 ± 30.122.23.80 ± 0.4854.20 ± 11.09162.3 ± 36.122.23.6651.98155.721.0this work
Non-Two-State Sequences
UGGCUA5.3649.70143.035.05.5436.1098.535.82.3224.6371.9–0.1104
CAUGUGC/7.70 ± 0.1449.10 ± 4.46133.5 ± 14.244.38.10 ± 0.1460.00 ± 11.18167.4 ± 35.645.15.9259.57173.033.8this work
GUGGUCG/7.93 ± 0.1856.21 ± 4.47155.7 ± 14.444.98.05 ± 0.1869.80 ± 6.64199.1 ± 18.143.97.6755.73154.943.6this work
GGUGUACC5.9465.80193.138.35.9949.60140.639.06.5461.72177.941.4104
GUAGCUGC6.5773.10214.440.96.4150.70142.841.68.1456.33155.351.3104
GAGGCGCGGAG/9.52 ± 0.24136.60 ± 11.90409.7 ± 37.743.98.28 ± 0.5659.27 ± 6.93164.4 ± 21.046.410.88111.46324.349.6this work
GCUUUGCGGAGC13.22 ± 0.35141.60 ± 7.01414.0 ± 21.554.410.39 ± 0.3482.58 ± 14.35232.7 ± 45.355.813.92129.51372.658.1this work
a

Experimental errors are listed for sequences melted in this study.

b

Listed in order of length of the oligoribonucleotide and then alphabetically for sequences of the same length. All non-self-complementary sequences have a slash and only one sequence shown. GU base pairs are underlined.

c

Calculated at a total strand concentration of 1 × 10–4 M.

Table 5 also lists duplexes that do not melt in a two-state manner. There are many possible reasons for this.(56, 63-65) The average difference between experimental and predicted TM for these sequences is 10.0 °C, while the predicted free energy is, on average, within 1.33 kcal/mol of the experimental free energy (Table 5). Evidently, the INN model may provide useful predictions for non-two-state sequences even though ΔH° from the van’t Hoff equation is erroneous.(64)

The Expanded Database Improves Predictions of Duplex Stability

Using the previous parameters,(12) the correlation coefficients between experimental values for ΔG°37, ΔH°, and ΔS° and those predicted for the 70 duplexes in Table 2 are 0.89, 0.86, and 0.85, respectively. Comparisons of the values of ΔG°37, ΔH°, and ΔS° of the 70 duplexes as predicted with the previous parameters,(12) and those in Table 3yielded, respectively, means of the differences of −0.36 kcal/mol, −1.75 kcal/mol, and −4.5 eu. The paired t-test gives t-values of −3.386, −2.528, and −2.257, respectively, which have absolute magnitudes greater than 1.995, indicating that the two sets of parameters differ with a significance level of 0.05 for 69 degrees of freedom.(61) Furthermore, the respective p-values of 1.2 × 10–3, 1.4 × 10–2, and 2.7 × 10–2 are less than 0.05. This again indicates that the new parameters predict the thermodynamics of RNA duplexes significantly differently from those published previously.(12)
Using the set of GU parameters in Table 3, the correlation coefficients between experimental values for ΔG°37, ΔH°, and ΔS° and those predicted for the 70 duplexes in Table 2 are 0.95, 0.89, and 0.87, respectively. Comparison of experimental values of ΔG°37, ΔH°, and ΔS° of the 70 duplexes and those predicted with the set of GU parameters in Table 3 yielded means of differences of −0.05 kcal/mol, 0.58 kcal/mol, and 2.1 eu and t-values of −0.544, 0.590, and 0.686, respectively, which have absolute magnitudes less than 1.995. The corresponding p-values of 0.59, 0.56, and 0.50 are greater than 0.05. These results show that the thermodynamic properties predicted with the new INN parameters are not significantly different from experiment.(61) The same analysis yielded means of differences of −0.41 kcal/mol, −1.17 kcal/mol, and −2.4 eu and t-values of −2.752, −1.096, and −0.736, with corresponding p-values of 7.6 × 10–3, 0.28, and 0.46 for ΔG°37, ΔH°, and ΔS°, respectively, when experimental properties were compared with predicted properties using previous parameters.(12) Evidently, the expanded database provides improved modeling of the thermodynamics of GU pairs.

The Nearest Neighbor Model Is Not Perfect

While the nearest neighbor model predicts well the ΔG°37 for most of the duplexes in Table 2, there are likely to be other terms that partially control stability. For example, there are four duplexes, r(GGCGUC)2, r(AGUCGAUU)2, r(UCACGUGG)2, and r(CCGAAUUUGG)2 with predicted ΔG°37 values not within 1.0 kcal/moland 20% of the 1/TM vs ln(CT/a) experimental values. No pattern is evident for these duplexes. A series of 1D spectra were acquired for r(CCGAAUUUGG)2 at different temperatures (Figure 1) because its predicted free energy is 1.5 kcal/mol more favorable at 37 °C than measured. These spectra show that the imino protons of all but U8, which is in the GU pair, and G10, which is in the terminal base pair disappear with each other, consistent with the duplex melting in a two-state manner. The results suggest that the nearest neighbor model does not include all factors that determine stabilities of duplexes with GU pairs.

Figure 1

Figure 1. 1D 1H imino spectra of r(CCGAAUUUGG)2 from 0 to 45 °C.

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The expanded database allows preliminary testing of models beyond the nearest neighbor model. For example, terminal GU pairs could be considered separately(43) and a base pair triplet model used for internal GU pairs. Comparison of measured values of ΔG°37 for terminal GU pairs with those predicted from the parameters in Table 3 give a standard deviation within 0.30 kcal/mol at 37 °C (Supporting Information). For the 16 triplets, 5′WGY/3′XUZ, with WX and YZ as Watson–Crick pairs, 12 measured ΔG°37(GU component) values are within 0.5 kcal/mol of the predicted values and the others within 1.0 kcal/mol (Supporting Information). The nearest neighbor model is apparently a reasonable approximation, and considerably more data would be required to develop a triplet model.
One clear exception to the nearest neighbor model is multiple terminal GU pairs.(43) Thus, the parameters in Table 3 cannot be used beyond the first terminal GU pair at a helix end. Parameters for additional terminal GU pairs have been published by Nguyen and Schroeder.(43)

Imino Proton NMR Spectra of Several Duplexes Are Consistent with the Expected Base Pairing

To check for expected base pairing, NMR imino proton spectra were measured for 12 duplexes. All had chemical shifts from 10 to 15 ppm (Figure 2). Chemical shifts for GH1 and UH3 of GU pairs were relatively upfield (10–12 ppm), consistent with expectations.(66) Chemical shifts for UH3 in AU pairs resonated between 13 and 15 ppm and GH1 in GC pairs resonated from 12 to 13.5 ppm, as expected.(67, 68) The absence of an imino peak for a terminal base pair in r(CUGGCUAG)2 indicates exchange with water. The G3-H1 and U7–H3 resonances of r(CUGGAUUCAG)2 appear to overlap, as evident by the presence of a single large peak. These chemical shift signatures show that the RNA sequences form the expected duplexes.

Figure 2

Figure 2. 1D imino NMR spectra for some RNA duplexes with GU pairs. Spectra were acquired at 0 °C for r(AGGCUU)2 and r(AUGCGU)2; 1 °C for r(AGUCGAUU)2; 5 °C for others.

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Discussion

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GU pairs are the most common non-Watson–Crick base pairs in RNA structures. Thus, the thermodynamics of GU pairs are important for finding regions of RNA that are structured,(1, 14-16, 69) predicting the secondary structure(12) or determining structure on the basis of chemical modification(8, 70) and/or NMR data.(71)
GU pairs can serve as binding sites for proteins or metal ions and participate in tertiary interactions.(72, 73) Thus, a better characterization of the thermodynamic properties of GU pairs can improve prediction of secondary and tertiary structure and help predict binding sites for metal ions and target sites for therapeutics. For example, GU pairs in group I introns can bind cations, including Mg2+, Co3+, and Os3+.(38, 40, 74, 75) Divalent metal ion binding by GU pairs, which have greater negative potential in the major groove than other base pairs, was postulated as important for activating RNA catalysis.(76) Divalent ions that interact with a GU pair help catalyze splicing by group I and group II introns(77-82) and cleavage by HDV ribozyme.(35) Metal ion binding with RNA neutralizes negative potential, which may promote higher order RNA folding.(75) The 5′GG/3′UU and 5′GU/3′UG motifs particularly contain greater negative potential in the major groove than their Watson–Crick counterparts.(83)

The Database of Sequences for Determining GU Thermodynamic Parameters Was Expanded

Not including sequences containing the 5′GGUC/3′CUGG motif, the database in Table 2 expands from 35 to 70 the duplexes used to fit nearest neighbor parameters for GU pairs. This expansion includes published data not included in the original database(43, 84-86) along with 29 new measurements (Table 2). Two of the original 35 duplexes were removed from the database because their melting temperatures were below 25 °C, which makes it difficult to analyze the melting curves. A third duplex, r(AUCUAGGU)2, was omitted because two-state melting could not be confirmed. The expanded database contains GU pairs flanked by Watson–Crick pairs in all possible orientations (Table 1). The new set of GU INN parameters were obtained with consideration for propagated errors from experiment and from Watson–Crick nearest neighbor parameters. Errors for the free energies of individual nearest neighbors were less than 0.2 kcal/mol for tandem GU pairs and 0.1 kcal/mol for other GU motifs. The 5′GG/3′UU motif, which was previously represented by a single sequence, was added to the fitting. The favorable free energy of −0.25 ± 0.16 kcal/mol for 5′GG/3′UU is in better agreement with the value of −0.5 kcal/mol used by Mathews et al.(12) to optimize secondary structure prediction than with the previous single experimental measurement of 0.47 kcal/mol.

GU Pairs Are Generally Less Stable than GC and AU Pairs

The free energies of formation for many of the duplexes with GU pairs (Table 2) can be compared with the free energies when the U or G of the GU pairs is replaced with a C or A, respectively, to form GC or AU pairs (Table 6). Because many of the latter duplexes terminated with a 3′ phosphate, the comparisons assume that the 3′ phosphate has negligible effect on ΔG°37 at 1 M NaCl.(87, 88) Duplexes containing GC pairs in place of GU pairs are more stable at 37 °C by 1.8 ± 0.8 kcal/mol per GU pair (Table 6). This is presumably due to the presence of an additional hydrogen bond in GC pairs and unfavorable backbone distortion due to GU pairs. Terminal substitutions all have a less than average effect while internal substitutions have a larger than average effect, as expected if backbone distortion is less important for a terminal GU.
Table 6. Free Energy Differences When GU Pairs Are Replaced with AU or GC Pairs
GC duplexrefΔG°37 GC duplexGU duplex.refΔG°37 GU duplexΔΔG°37 per GU pair (kcal/mol)
CCGCGG119.84CUGCGG1014.312.77
CGGCCGp1109.90CGGCUG1015.552.18
CGGCCGp1109.90UGGCCGp1028.560.67
CUGCAGp1117.11UUGCAG434.201.46
GCCGGCp11011.20GCCGGUp1029.171.02
GGCGCCp11211.33GGCGCU1028.421.46
GGCGCCp11211.33GGCGUC1014.673.33
GUGCAC1117.65GUGCAU435.101.28
AUGCGCAUp10110.17AUGCGUAUp1015.272.45
CAUGCAUGp1139.67UAUGCAUGp1066.441.62
GAUGCAUCp11310.12GAUGCAUUp1066.821.65
GCAGCUGC11413.87GCAGCUGUthis work10.301.79
average 1.80 ± 0.76
AU duplexrefΔG°37 AU duplexGU duplex.refΔG°37 GU duplexΔΔG°37 per GU pair (kcal/mol).
ACCGGUp1158.51GCCGGUp1029.17–0.33
AGCGCU1127.99GGCGCU1028.42–0.22
CAGCUGp1116.68CGGCUG1015.550.57
CUGCAGp1117.11CUGCGG1014.311.40
GACGUC1167.35GGCGUC1014.671.34
UCCGGAp887.99UCCGGGp1027.440.28
CUCACUC/119.71CUCGCUC/1177.781.93
AAUGCAUUp1137.18GAUGCAUUp1066.820.18
AUACGUAU1016.53AUGCGUAUp1015.270.63
AUGCGCAUp10110.17AUGCGCGUp1019.310.43
UAUGCAUAp1137.27UAUGCAUGp1066.440.42
Average 0.60 ± 0.70
The effect of replacing GU with GC pairs can be compared to replacing AU pairs with GC pairs (Supporting Information). On average, replacing an AU pair with a GC pair stabilized a duplex by 1.5 ± 0.4 kcal/mol per AU pair. In this case, there was no apparent difference between terminal and internal substitutions.
Duplexes containing AU pairs in place of GU pairs are more stable at 37 °C by 0.6 ± 0.7 kcal/mol per GU pair (Table 6). While the difference is zero within the standard deviation, in only 2 of 11 cases is the GU duplex more stable than the AU duplex and in both cases the difference is within the experimental error of 4%.
Unlike terminal AU pairs, no penalty for terminal GU pairs is required to account for base pair composition. The terminal AU penalty of 0.45 kcal/mol at 37 °C was considered to account for numbers of base pairing hydrogen bonds.(11) Thus, the penalty for terminal GU pairs was assumed to be equal to that of AU pairs,(12) consistent with wobble GU pairs at the end of a helix having two hydrogen bonds.(89) When the terminal GU parameter was included in the reparameterization of GU nearest neighbor thermodynamic parameters, the free energy of each nearest neighbor parameter differed by no more than 0.01 kcal/mol from that calculated without it (Table 2 and Supporting Information). The lack of a terminal GU penalty may arise from the flexibility of a terminal GU pair which allows optimization of hydrogen bonding and stacking interactions without incurring the energetic penalty associated with an interior GU distorting the backbone.(43) For example, even for an internal GU pair, optimal stability may be found with only one hydrogen bond due to stacking energies.(90, 91) Thus, flexibility of terminal GU’s may compensate for the difference between the free energy of formation of two and three hydrogen bonds in GU and GC pairs, respectively.

Tandem GU Pairs Have Structural Features That Correlate with Their Thermodynamic Properties

With the exception of 5′GGUC/3′CUGG, the 5′UG/3′GU motif is more stable than 5′GU/3′UG (Table 3). Available structures show that 5′UG/3′GU contains interstrand stacking between the guanines,(90-93) whereas 5′GU/3′UG does not.(91, 94, 95) The favorability of the 5′UG/3′GU motif relative to the 5′GU/3′UG motif is consistent with molecular dynamics (MD) simulations(91) that predict a one hydrogen bond GU pair(90) predominates in duplexes containing the 5′GU/3′UG motif while a two hydrogen bond model predominates in duplexes containing the 5′UG/3′GU motif. There is also less overlap of negative potentials in 5′UG/3′GU than in 5′GU/3′UG.(95) In two different sequences containing the 5′UG/3′GU motif, there is also intrastrand stacking between each GU pair and its Watson–Crick neighbors.(92, 93) By comparison, the 5′GU/3′UG motif contains less overlap between the GU pairs and Watson–Crick purine neighbors, but has intrastrand stacking between the tandem GU pairs.(94) Furthermore, the 5′UG/3′GU motif preserves the A-form of RNA more than 5′GU/3′UG.(96)
The 5′GGUC/3′CUGG motif is an exception to the above generalizations. NMR spectra and modeling indicate that the GU pairs of r(GAGGUCUC)2 contain two hydrogen bonds,(52) whereas the GU pairs in r(GGCGUGCC)2 contain only one hydrogen bond.(90) This difference would contribute to the favorable free energy of 5′GGUC/3′CUGG compared to that of 5′GU/3′UG in other contexts, such as 5′CGUG/3′GUGC. Pan et al. saw similar hydrogen-bonding scenarios in MD simulations.(91) Additional stability for the 5′GGUC/3′CUGG motif may also be conferred from less overlap of its negative electrostatic potentials between a GC and GU pair than for its related motif, 5′CGUG/3′GUGC.(52) These patterns may explain the poor fit of nearest neighbor parameters for the 5′GU/3′UG motif when duplexes containing the 5′GGUC/3′CUGG motif are included in the fit. Alternatively, the extra stability of the 5′GGUC/3′CUGG motif over 5′CGUG/3′GUGC may arise from poor cross-strand overlap between the U in a GU pair and the C in its neighboring GC pair in 5′CGUG/3′GUGC.(97) Stacking interactions alone do not contribute to the stability of nearest neighbor motifs comprised of the same base pairs, however, as evident from the comparable stability of 5′UG/3′GU and 5′GG/3′UU. This contrasts with the expectation that the free energy of 5′GG/3′UU is between the other tandem GU motifs because its base stacking is intermediate among them.(98) Understanding the interactions responsible for the observed sequence dependence of thermodynamics presents a challenge to computational chemists.

GU Pairs of RNA Are More Stable than GT Pairs of DNA

Comparison of ΔG°37 values for GT nearest neighbors in DNA(74) with those measured for GU nearest neighbors show that GT nearest neighbors are on average 0.84 ± 0.36 kcal/mol less stable than their GU counterparts. The extra stability of GU relative to GT is also evident from comparisons of ΔG°37 (GU or GT component) for duplexes containing comparable triplet motifs (Table 7). This may reflect a possible hydrogen bond between the amino group of guanine and the O2′ of uracil,(99) which is not possible with DNA. MD simulations utilizing residual dipolar coupling (RDC) restraints suggest that the 5′TG/3′GT motif contains a bifurcated hydrogen bond(100) similar to that in the 5′GU/3′UG motif.(90, 91) Another difference between GT and GU is that the 5′GGTC/3′CTGG motif fits the nearest neighbor parameters for the 5′GT/3′TG motif better than their respective uracil-substituted RNA motifs.(74) Consistent with the relative stabilities of GT and GU nearest neighbors, component free energies of GT pairs in duplexes are consistently less favorable than those of GU pairs flanked by the same Watson–Crick pairs (Table 7).
Table 7. Component Free Energies of GU and GT Pairsa
sequencesbΔG°37(1/TM vs ln(CT/a)) (kcal/mol)experimental ΔG°37(component) (kcal/mol)predicted ΔG°37(component)c(kcal/mol)d
GCGUGC–5.11–2.79–1.78
GACCGTGCAC/–7.17–0.400.20
AUGCGUAUc–5.27–3.07–2.54
CCATGCGTAACG/–8.94–0.90–0.30
CTTGCATGTAAGc,e–6.10–0.55–0.15
CUCGGCUC/–8.22–3.45–3.65
GACGTTGGAC/–7.91–1.40–0.30
CUGGCUAG–7.10–4.04–4.32
CTTGGATCTAAG–5.89–1.200.20
GAGUGCUC–9.40–5.06–4.87
GGAGTGCTCC–7.66–2.20–0.70
GCAGUUGC–5.900.640.02
GGAGUUCC–6.430.270.02
GGCAGTTCGC/–6.871.402.60
GCAUGUGC–8.40–1.86–2.37
GGAUGUCC–8.39–1.69–2.37
GCGATGTCGCe–7.980.100.70
CAGUCGAUUGc–8.70–0.97–1.25
GTACAGTGATC/–7.78–1.200.80
CGAGTCGATTCGc,e–7.710.350.80
GAGAGCUUUC–8.82–1.06–1.72
CGAGACGTTTCG–6.961.702.10
GAGGAUCUUCc–9.83–1.93–2.12
CATGAGGCTAC/–8.57–0.900.40
GAGUGGAGAG/–9.87–0.78–1.15
GACTGGAGAG/e–4.610.301.50
GUGAAUUUAC–4.78–1.86–1.80
GUUAGCUGAC–8.60–1.06–1.80
CGTGACGTTACG–8.190.701.50
CGTTACGTGACG–7.861.101.50
GUGUGCAUAC–8.90–1.30–2.58
CGTGTCGATACG–8.420.201.00
CGTGTCTAGATACGe–9.400.201.00
a

Data for DNA duplexes were referenced from ref 74.

b

Listed in order of length of the oligoribonucleotide and then alphabetically for sequences of the same length. All nonself-complementary sequences have a slash and only one sequence shown. GU and GT base pairs are underlined.

c

Component free energies were divided by 2.

d

Calculated with free energies in Table 3.

e

Marginally non-two-state.(74)

Supporting Information

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(I) Thermodynamic parameters for duplex formation of Watson–Crick sequences. (II) Experimental thermodynamic parameters and error limits for newly measured sequences. (III) Component free energies and enthalpies of GU pairs. (IV) Free energies of doublets and triplets containing GU pairs calculated as component ΔG°37 of their sequences. (V) Free energy differences between sequences where GC pair(s) were replaced by AU pair(s). (VI) INN parameters for GU pairs calculated with a separate term for terminal GU pairs. (VII) Probability density function of the Student’s t-distribution for each INN motif with a separate parameter for terminal GU pairs. (VIII) 2D NOESY spectra for r(AGGCUU)2 showing H2′, H1′, and H6/H8 regions. (IX) 2D NOESY spectra for r(AUGCGU)2 showing H2′, H1′, and H6/H8 regions. (X) Desalting procedure for oligoribonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

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Acknowledgment

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The authors thank Zhenjiang Xu for suggesting the paired t-test and Dr. Susan Schroeder for comments on the manuscript.

Abbreviations

1D

one-dimensional

2D

two-dimensional

HIV-1

human immunodeficiency virus-1

INN

individual nearest neighbor

MD

molecular dynamics

NN

nearest neighbor

NOESY

nuclear Overhauser effect spectroscopy

PDF

probability density function

RDC

residual dipolar coupling

SVD

singular value decomposition

TOCSY

total correlation spectroscopy

WC

Watson–Crick

References

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This article references 117 other publications.

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    BACKGROUND: Non-coding RNAs (ncRNAs) have a multitude of roles in the cell, many of which remain to be discovered. However, it is difficult to detect novel ncRNAs in biochemical screens. To advance biological knowledge, computational methods that can accurately detect ncRNAs in sequenced genomes are therefore desirable. The increasing number of genomic sequences provides a rich dataset for computational comparative sequence analysis and detection of novel ncRNAs. RESULTS: Here, Dynalign, a program for predicting secondary structures common to two RNA sequences on the basis of minimizing folding free energy change, is utilized as a computational ncRNA detection tool. The Dynalign-computed optimal total free energy change, which scores the structural alignment and the free energy change of folding into a common structure for two RNA sequences, is shown to be an effective measure for distinguishing ncRNA from randomized sequences. To make the classification as a ncRNA, the total free energy change of an input sequence pair can either be compared with the total free energy changes of a set of control sequence pairs, or be used in combination with sequence length and nucleotide frequencies as input to a classification support vector machine. The latter method is much faster, but slightly less sensitive at a given specificity. Additionally, the classification support vector machine method is shown to be sensitive and specific on genomic ncRNA screens of two different Escherichia coli and Salmonella typhi genome alignments, in which many ncRNAs are known. The Dynalign computational experiments are also compared with two other ncRNA detection programs, RNAz and QRNA. CONCLUSION: The Dynalign-based support vector machine method is more sensitive for known ncRNAs in the test genomic screens than RNAz and QRNA. Additionally, both Dynalign-based methods are more sensitive than RNAz and QRNA at low sequence pair identities. Dynalign can be used as a comparable or more accurate tool than RNAz or QRNA in genomic screens, especially for low-identity regions. Dynalign provides a method for discovering ncRNAs in sequenced genomes that other methods may not identify. Significant improvements in Dynalign runtime have also been achieved.
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    Many non-coding RNA genes and cis-acting regulatory elements of mRNAs contain RNA secondary structures that are critical for their function. Such functional RNAs can be predicted on the basis of thermodynamic stability and evolutionary conservation. We present a web server that uses the RNAz algorithm to detect functional RNA structures in multiple alignments of nucleotide sequences. The server provides access to a complete and fully automatic analysis pipeline that allows not only to analyze single alignments in a variety of formats, but also to conduct complex screens of large genomic regions. Results are presented on a website that is illustrated by various structure representations and can be downloaded for local view. The web server is available at: rna.tbi.univie.ac.at/RNAz.
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    Biochemistry (1992), 31 (6), 1610-21CODEN: BICHAW; ISSN:0006-2960.
    The structure of helix I of the 5 S rRNA from E. coli has been detd. using a nucleolytic digest fragment of the intact mol. The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5 S rRNA, contains a G-U (guanine-uracil) pair and has unpaired bases at its termini. Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling const. information have been used to calc. structural models for it using the full relaxation matrix algorithm of the mol. dynamics program XPLOR. Helix I has A-type helical geometry, as expected. Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair on its 3' side. This stacking pattern maximized interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G. These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe and A-form DNA. The configurations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5 S RNA, are less well-detd., and their sugar puckers are intermediate between C2'- and C3'-endo, on av.
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  19. 19
    Szymański, M., Barciszewska, M. Z., Erdmann, V. A., and Barciszewski, J. (2000) An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs Mol. Biol. Evol. 17, 1194– 1198
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    19
    An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs
    Szymanski, Maciej; Barciszewska, Miroslawa Z.; Erdmann, Volker A.; Barciszewski, Jan
    Molecular Biology and Evolution (2000), 17 (8), 1194-1198CODEN: MBEVEO; ISSN:0737-4038. (Society for Molecular Biology and Evolution)
    The structure-function relationship in RNA mols. is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an anal. of the distribution of G-U pairs in eukaryotic 5S rRNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.
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  20. 20
    Sprinzl, M. and Vassilenko, K. S. (2005) Compilation of tRNA sequences and sequences of tRNA genes Nucleic Acids Res. 33, D139– D140
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    Compilation of tRNA sequences and sequences of tRNA genes
    Sprinzl, Mathias; Vassilenko, Konstantin S.
    Nucleic Acids Research (2005), 33 (Database), D139-D140CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Maintained at the Universitaet Bayreuth, Bayreuth, Germany, the Compilation of tRNA Sequences and Sequences of tRNA Genes is accessible at the URL http://www.tRNA.uni-bayreuth.de with mirror site located at the Institute of Protein Research, Pushchino, Russia (http://alpha.protres.ru/trnadbase). The compilation is a searchable, periodically updated database of currently available tRNA sequences. The present version of the database contains a new Genomic tRNA compilation including the sequences of tRNA genes from genomic sequences published up to July 2003. It consists of about 5800 tRNA gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya. The former Compilation of tRNA Genes (up to the end of 1998) and the updated compilation tRNA Sequences (561 entries) are also supported by the new software. The database can be explored by using multiple search criteria and sequence templates. The database provides a service that allows to obtain statistical information on the occurrences of certain bases at given positions of the tRNA sequences. This allows phylogenic studies and search for identity elements in respect to interactions of tRNAs with various enzymes.
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  21. 21
    Limmer, S., Reif, B., Ott, G., Arnold, L., and Sprinzl, M. (1996) NMR evidence for helix geometry modifications by a G-U wobble base pair in the acceptor arm of E-coli tRNA(Ala) FEBS Lett. 385, 15– 20
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    NMR evidence for helix geometry modifications by a G-U wobble base pair in the acceptor arm of E. coli tRNAAla
    Limmer, Stefan; Reif, Bernd; Ott, Geunther; Arnold, Lubos; Sprinzl, Mathias
    FEBS Letters (1996), 385 (1,2), 15-20CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
    A ribooligonucleotide duplex representing the acceptor stem of Escherichia coli tRNAAla with a G3-U70 wobble base pair, which is the main identity element for the recognition by the alanine-tRNA synthetase, has been characterized by 2D-NMR, as having two sequence variants with a regular Watson-Crick G3-C70 and an I3-U70 wobble pair, resp. As compared to a regular A-RNA, the G-U base pair gives rise to variations of the local helix geometry which are reflected in distinct local chem. shift changes. Structural differences between the duplex possessing an I3-U70 base pair and the wild-type G3-U70 sequence have also been found. The nucleotides in the ubiquitous single-stranded NCCA terminus display a surprisingly high degree of stacking order, esp. between A73, C74, and C75.
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    Hou, Y. M. and Schimmel, P. (1988) A simple structural feature is a major determinant of the identity of a transfer RNA Nature 333, 140– 145
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    A simple structural feature is a major determinant of the identity of a transfer RNA
    Hou, Ya Ming; Schimmel, Paul
    Nature (London, United Kingdom) (1988), 333 (6169), 140-5CODEN: NATUAS; ISSN:0028-0836.
    Anal. of a series of mutants of an Escherichia coli alanine tRNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine tRNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific tRNA.
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    McClain, W. H. and Foss, K. (1988) Changing the identity of a transfer RNA by introducing a G-U wobble pair near the 3′ acceptor end Science 240, 793– 796
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    Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end
    McClain, William H.; Foss, K.
    Science (Washington, DC, United States) (1988), 240 (4853), 793-6CODEN: SCIEAS; ISSN:0036-8075.
    Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of tRNA has remained unknown. To investigate the identity of a tRNA, the nucleotides at 3 computer-identified positions in tRNAPhe were replaced with the corresponding nucleotides from tRNAAla. The identity of the resulting tRNA, when examd. as an amber suppressor in Escherichia coli, was that of tRNAAla.
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  24. 24
    Mueller, U., Schubel, H., Sprinzl, M., and Heinemann, U. (1999) Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G·U wobble base pair at 1.16 angstrom resolution RNA 5, 670– 677
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    24
    Crystal structure of acceptor stem of tRNAAla from Escherichia coli shows unique G·U wobble base pair at 1.16 Å resolution
    Mueller, Uwe; Schubel, Harald; Sprinzl, Mathias; Heinemann, Udo
    RNA (1999), 5 (5), 670-677CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    The acceptor stem of Escherichia coli tRNAAla, rGGGGCUA·rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3-U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the crit. feature recognized by the synthetase. The authors here report the structure anal. of ALAwt at near-at. resoln. using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses. The two independent mols. in the asym. unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALAc70) detd. at 1.4 Å exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. The authors suggest that a highly conserved hydration pattern in both grooves around the G3·U70 wobble base pair may be functionally significant.
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    White, S. A. and Li, H. (1996) Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition RNA 2, 226– 234
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    Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition
    White, Susan A.; Li, Hu
    RNA (1996), 2 (3), 226-34CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    Yeast ribosomal protein L32, RPL32, specifically represses splicing by binding to a purine-rich asym. loop adjacent to the 5' splice site of its own transcript. A potential G:U pair closes the internal loop and the goal of the present study is to understand what features of the putative G:U pair are recognized by RPL32. Two RNA oligomers contg. 10 and 13 nt were annealed to form a bimol. stem-loop-stem protein-binding site. Protein binding to each of 16 sequence variants was examd. using electrophoretic bandshift and filter-binding expts. The protein binds to only the duplex RNA and not to the individual oligomers, and the G:U pair is crit. for full-strength binding. Mutation studies show that the duplex having a G:U has the highest protein affinity (Kd = 10 nM), followed by RNAs bearing G:A, C:C, U:A, U:C, or G:G. Duplexes contg. the other possible pairs bind very weakly and Watson-Crick pairing does not favor protein binding. The G of the G:U is required for strong protein binding, but replacement by inosine reduces binding only modestly. Therefore, the minor groove guanine amino group is not a key protein recognition element. Both nucleotides of the pair influence the binding strength, but their contributions are in general not additive. These data imply that the G:U is probably paired and influences binding indirectly through its effect on the conformation of the RNA.
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    Reyes, J. L., Gustafson, E. H., Luo, H. R., Moore, M. J., and Konarska, M. M. (1999) The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site RNA 5, 167– 179
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    26
    The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site
    Reyes, Jose L.; Gustafson, E. Hilary; Luo, Hongbo R.; Moore, Melissa J.; Konarska, Maria M.
    RNA (1999), 5 (2), 167-179CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    A U5 snRNP protein, hPrp8, forms a UV-induced crosslink with the 5' splice site (5'SS) RNA within splicing complex B assembled in trans- as well as in cis-splicing reactions. Both yeast and human Prp8 interact with the 5'SS, branch site, polypyrimidine tract, and 3'SS during splicing. To begin to define functional domains in Prp8 we have mapped the site of the 5'SS crosslink within the hPrp8 protein. Immunopptn. anal. limited the site of crosslink to the C-terminal 50-60-kDa segment of hPrp8. In addn., size comparison of the crosslink-contg. peptides generated with different proteolytic reagents with the pattern of fragments predicted from the hPrp8 sequence allowed for mapping of the crosslink to a stretch of five amino acids in the C-terminal portion of hPrp8 (positions 1894-1898). The site of the 5'SS:hPrp8 crosslink falls within a segment spanning the previously defined polypyrimidine tract recognition domain in yPrp8, suggesting that an overlapping region of Prp8 may be involved both in the 5'SS and polypyrimidine tract recognition events. In the context of other known interactions of Prp8, these results suggest that this protein may participate in formation of the catalytic center of the spliceosome.
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  27. 27
    Leung, S. S. and Koslowsky, D. J. (2001) Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of an mRNA bound to its cognate gRNA RNA 7, 1803– 1816
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    Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of an mRNA bound to its cognate gRNA
    Leung, Sheldon S.; Koslowsky, Donna J.
    RNA (2001), 7 (12), 1803-1816CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    Posttranscriptional editing of trypanosome mitochondrial mRNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5' anchor, whereas its 3' U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examd. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examd. using soln.-probing techniques. The mRNA/gRNA crosslinked mols. are efficient substrates for gRNA-directed cleavage. In addn., when the cleavage assay is performed in the presence or absence of addnl. UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the mols. in a biol. relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide addnl. support for the predicted secondary structure of interacting gRNA/mRNA pairs.
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  28. 28
    Mooers, B. H. M. and Singh, A. (2011) The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate RNA 17, 1870– 1883
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    28
    The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate
    Mooers, Blaine H. M.; Singh, Amritanshu
    RNA (2011), 17 (10), 1870-1883CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 5' end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 3' ends of most guide RNAs have ∼15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G·U wobble base pairs. To gain insights into the structure of the U-helix, we crystd. 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 5'-AGA-3'/5'-UUU-3' base-pair triple. The fusion of two identical U-helixes head-to-head promoted crystn. We obtained X-ray diffraction data with a resoln. limit of 1.37 Å. The U-helix had low and high twist angles before and after each G·U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins.
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  29. 29
    Lu, K., Heng, X., Garyu, L., Monti, S., Garcia, E. L., Kharytonchyk, S., Dorjsuren, B., Kulandaivel, G., Jones, S., Hiremath, A., Divakaruni, S. S., LaCotti, C., Barton, S., Tummillo, D., Hosic, A., Edme, K., Albrecht, S., Telesnitsky, A., and Summers, M. F. (2011) NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging Science 334, 242– 245
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    29
    NMR detection of structures in the HIV-1 5'-leader RNA that regulate genome packaging
    Lu, Kun; Heng, Xiao; Garyu, Lianko; Monti, Sarah; Garcia, Eric L.; Kharytonchyk, Siarhei; Dorjsuren, Bilguujin; Kulandaivel, Gowry; Jones, Simonne; Hiremath, Atheeth; Divakaruni, Sai Sachin; LaCotti, Courtney; Barton, Shawn; Tummillo, Daniel; Hosic, Azra; Edme, Kedy; Albrecht, Sara; Telesnitsky, Alice; Summers, Michael F.
    Science (Washington, DC, United States) (2011), 334 (6053), 242-245CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a NMR approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are crit. for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.
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  30. 30
    Knitt, D. S., Narlikar, G. J., and Herschlag, D. (1994) Dissection of the role of the conserved G·U pair in group I RNA self-splicing Biochemistry 33, 13864– 13879
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    30
    Dissection of the Role of the Conserved G·U Pair in Group I RNA Self-Splicing
    Knitt, Deborah S.; Narlikar, Geeta J.; Herschlag, Daniel
    Biochemistry (1994), 33 (46), 13864-79CODEN: BICHAW; ISSN:0006-2960.
    Phylogenetic conservation among >100 group I introns and previous in vitro studies have implicated a G· U pair as defining the 5'-splice site for exon ligation. The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon. The authors now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA. The following results indicate that interactions with this G·U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chem. step. The oligonucleotide, CCCUCU, binds to the ribozyme ∼20-fold stronger than CCCUCC despite the fact that the U-contg. oligonucleotide forms an ∼5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG. This and two independent exptl. observations indicate that the G·U pair contributes ∼100-fold (3 kcal/mol, 50°) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The ∼50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G·U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites. Comparison of the rates of the chem. cleavage step with G·U vs. G·C suggests that the G·U pair contributes ∼10-fold to the chem. step. It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chem. transition state via an intramol. hydrogen bond. Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent x-ray crystallog. structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water mol., thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion. Finally, kinetic and thermodn. evidence for the formation of a G·C+ wobble pair is presented. The tertiary energy of recognition of the G·U wobble pair appears to be sufficient to perturb the pKa of C to favor a G·C+ wobble pair instead of the Watson-Crick pair, despite the loss of a hydrogen bond in the base pair. This provides an example of RNA tertiary structure detg. secondary structure.
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  31. 31
    Pyle, A. M., Moran, S., Strobel, S. A., Chapman, T., Turner, D. H., and Cech, T. R. (1994) Replacement of the conserved G·U with a G-C pair at the cleavage site of the tetrahymena ribozyme decreases binding, reactivity, and fidelity Biochemistry 33, 13856– 13863
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    31
    Replacement of the Conserved G·U with a G-C Pair at the Cleavage Site of the Tetrahymena Ribozyme Decreases Binding, Reactivity, and Fidelity
    Pyle, Anna Marie; Moran, Sean; Strobel, Scott A.; Chapman, Teresa; Turner, Douglas H.; Cech, Thomas R.
    Biochemistry (1994), 33 (46), 13856-63CODEN: BICHAW; ISSN:0006-2960.
    There is a phylogenetically conserved G·U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. The authors have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G·U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42°C was measured by native gel electrophoresis and equil. dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G·U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G·U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position. Taken together, the results suggest that a G-C pair at the ribozyme cleavage site makes docking of the guide-sequence-substrate helix into the catalytic site less favorable than a G·U pair. The resulting consequences of weaker binding, slower reaction, and reduced cleavage fidelity provide a rationale for the phylogenetic conservation of the G·U.
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  32. 32
    Strobel, S. A. and Cech, T. R. (1995) Minor groove recognition of the conserved G·U pair at the tetrahymena ribozyme reaction site Science 267, 675– 679
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    32
    Minor groove recognition of the conserved G·U pair at the Tetrahymena ribozyme reaction site
    Strobel, Scott A.; Cech, Thomas R.
    Science (Washington, D. C.) (1995), 267 (5198), 675-9CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The guanine-uracil (G·U) base pair that helps to define the 5'-splice site of group I introns is phylogenetically highly conserved. In such a wobble base pair, G makes 2 H-bonds with U in a geometry shifted from that of a canonical Watson-Crick pair. The contribution made by individual functional groups of the G.U pair in the context of the Tetrahymena ribozyme was examd. by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex. The substitutions demonstrate that the exocyclic amine of G, when presented on the minor groove surface by the wobble base pair conformation, contributes substantially (2 kcal.mole-1) to binding by making a tertiary interaction with the ribozyme active site. It contributes addnl. to transition state stabilization. The ribozyme active site also makes tertiary contacts with a tripod of 2'-hydroxyls on the minor groove surface of the splice site helix. This suggests that the ribozyme binds the duplex primarily in the minor groove. Alanyl-aminoacyl-tRNA synthetase recognizes the exocyclic amine of an invariant G.U pair and contacts a similar array of 2'-hydroxyls when binding the tRNAAla acceptor stem, providing an unanticipated parallel between protein-RNA and RNA-RNA interactions.
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  33. 33
    Strobel, S. A. and Cech, T. R. (1996) Exocyclic amine of the conserved G·U pair at the cleavage site of the Tetrahymena ribozyme contributes to 5′-splice site selection and transition state stabilization Biochemistry 35, 1201– 1211
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    33
    Exocyclic Amine of the Conserved G·U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and Transition State Stabilization
    Strobel, Scott A.; Cech, Thomas R.
    Biochemistry (1996), 35 (4), 1201-11CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    A phylogenetically conserved guanine·uracil (G·U) pair defines the 5'-exon/intron boundary of precursor RNAs contg. group I introns. In this wobble base pair, the G forms two hydrogen bonds with U in a base pairing geometry shifted from that of a canonical Watson-Crick pair. On the basis of thermodn. measurements of synthetic base pair analogs (inosine, diaminopurine riboside, guanosine, or adenosine paired with U, C, or isocytidine) in place of the G·U pair, the authors have previously reported that the N2 exocyclic amine of the G is important for docking the 5'-exon into the active site of the Tetrahymena ribozyme [Strobel, S. A., & Cech, T. R. (1995) Science 267, 675-679]. Here the authors describe kinetic characterization of ribozyme-substrate combinations contg. the same series of analogs. By measuring the rate consts. of 5'-exon miscleavage (cleavage at incorrect phosphates), the authors demonstrate that the 5'-exon/intron boundary is primarily defined by the exocyclic amine of the G. The amine makes its contribution (2.5 kcal·mol-1) in the context of all three wobble pairs tested but fails to make a significant contribution (<0.8 kcal·mol-1) when presented in a Watson-Crick base pairing geometry. The authors also demonstrate that the exocyclic amine makes a modest contribution to chem. transition state stabilization (1.0 kcal·mol-1 relative to an inosine-U pair). The majority of this transition state contribution (0.7 kcal·mol-1) is independent of that contributed by the 2'-hydroxyl of the neighboring U. This argues against the model in which substantial transition state stabilization is derived from a water mol. bridging between the exocyclic amine of G and the 2'-hydroxyl of U. Instead it suggests that the tertiary interaction between the exocyclic amine and its hydrogen bonding partner in the active site is slightly improved during the chem. transition. The authors conclude that the exocyclic amine of G is the primary contributor to many characteristics of reactivity that have been ascribed to the conserved G·U pair, including stabilization of the chem. transition state and definition of the 5'-exon/intron boundary.
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  34. 34
    Šponer, J., Šponer, J. E., Petrov, A. I., and Leontis, N. B. (2010) Quantum chemical studies of nucleic acids: Can we construct a bridge to the RNA structural biology and bioinformatics communities? J. Phys. Chem. B 114, 15723– 15741
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    Quantum Chemical Studies of Nucleic Acids: Can We Construct a Bridge to the RNA Structural Biology and Bioinformatics Communities?
    Sponer, Jiri; Sponer, Judit E.; Petrov, Anton I.; Leontis, Neocles B.
    Journal of Physical Chemistry B (2010), 114 (48), 15723-15741CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    A review. In this feature article, the authors provide a side-by-side introduction for 2 research fields: quantum chem. calcns. of mol. interaction in nucleic acids and RNA structural bioinformatics. The authors' main aim is to demonstrate that these research areas, while largely sepd. in contemporary literature, have substantial potential to complement each other that could significantly contribute to the authors' understanding of the exciting world of nucleic acids. The authors identify research questions amenable to the combined application of modern ab initio methods and bioinformatics anal. of exptl. structures while also assessing the limitations of these approaches. The ultimate aim is to attain valuable physicochem. insights regarding the nature of the fundamental mol. interactions and how they shape RNA structures, dynamics, function, and evolution.
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    Chen, J.-H., Gong, B., Bevilacqua, P. C., Carey, P. R., and Golden, B. L. (2009) A catalytic metal ion interacts with the cleavage site G·U wobble in the HDV ribozyme Biochemistry 48, 1498– 1507
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    A Catalytic Metal Ion Interacts with the Cleavage Site G·U Wobble in the HDV Ribozyme
    Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.
    Biochemistry (2009), 48 (7), 1498-1507CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The hepatitis delta virus (HDV) ribozyme self-cleaves by a chem. mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochem. studies from several labs. have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted >2 pH units toward neutrality, while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been obsd. in high-resoln. crystal structures of various reaction states and mutants. Herein, we use soln. kinetic expts. and parallel Raman crystallog. studies to examine the effects of pH on the rate and Mg2+ binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G·U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochem. data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochem. data.
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  36. 36
    Chen, J.-H., Yajima, R., Chadalavada, D. M., Chase, E., Bevilacqua, P. C., and Golden, B. L. (2010) A 1.9 Å crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage Biochemistry 49, 6508– 6518
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    36
    A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage
    Chen, Jui-Hui; Yajima, Rieko; Chadalavada, Durga M.; Chase, Elaine; Bevilacqua, Philip C.; Golden, Barbara L.
    Biochemistry (2010), 49 (31), 6508-6518CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pKa shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg2+ ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochem.; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystd. and detd. the structure of the HDV ribozyme bound to an inhibitor RNA contg. a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg2+ ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg2+ ion is also found within the active site where it interacts with a newly resolved G·U reverse wobble. Although the inhibitor exhibits crystallog. disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand obsd. in the hammerhead ribozyme. This model suggests that the pro-RP oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg2+ ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.
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    Keel, A. Y., Rambo, R. P., Batey, R. T., and Kieft, J. S. (2007) A general strategy to solve the phase problem in RNA crystallography Structure 15, 761– 772
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    A general strategy to solve the phase problem in RNA crystallography
    Keel, Amanda Y.; Rambo, Robert P.; Batey, Robert T.; Kieft, Jeffrey S.
    Structure (Cambridge, MA, United States) (2007), 15 (7), 761-772CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    X-ray crystallog. of biol. important RNA mols. has been hampered by tech. challenges, including finding heavy-atom derivs. to obtain high-quality exptl. phase information. Existing techniques have drawbacks, limiting the rate at which important new structures are solved. To address this, the authors have developed a reliable means to localize heavy atoms specifically to virtually any RNA. By solving the crystal structures of thirteen variants of the G·U wobble pair cation binding motif, the authors have identified a version that when inserted into an RNA helix introduces a high-occupancy cation binding site suitable for phasing. This "directed soaking" strategy can be integrated fully into existing RNA crystallog. methods, potentially increasing the rate at which important structures are solved and facilitating routine solving of structures using Cu-Kα radiation. This method already has been used to solve several crystal structures.
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    Kieft, J. S. and Tinoco, I. (1997) Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine Structure 5, 713– 721
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    Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine
    Kieft, Jeffrey S.; Tinoco, Ignacio, Jr.
    Structure (London) (1997), 5 (5), 713-721CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
    Solvated metal ions are crit. for the proper folding and function of RNA. Despite the importance of these ions, the details of specific metal ion-RNA interactions are poorly understood. The crystal structure of a group I intron ribozyme domain characterized several metal-binding sites in the RNA with osmium (III) hexammine bound in the major groove. A corresponding method for locating and characterizing metal-binding sites of RNA in soln. is of obvious interest. NMR should be ideal for localizing metal hexammine ions bound to the RNA because of the large concn. of protons around the metal center. We have solved the soln. structure of the P5b stem loop from a group I intron ribozyme bound to a cobalt (III) hexammine ion. The location of the ion is precisely detd. by intermol. nuclear Overhausser effect cross-peaks between the cobalt (III) hexammine protons and both exchangeable and non-exchangeable RNA protons in the major groove. The binding site consists of tandem G-U base pairs in a sequence of four consecutive G residues ending in a GAAA tetraloop, as originally identified in the crystal structure. The edges of the bases in the major groove present an electrostatically neg. face and a variety of hydrogen-bond acceptors for the cobalt (III) hexammine ion. The metal ion ligand is bound near the guanosine nucleotides of the adjacent G-U base pairs, where it makes hydrogen bonds with the N7 and carbonyl groups of both guanines. The carbonyl groups of the uracil residues add to the neg. surface of the binding pocket, but do not form hydrogen bonds with the hexammine. Addnl. hydrogen bonds form with other guanine residues of the GGGG sequence. The structure of the binding site does not change significantly on binding the cobalt (III) hexammine. The structure of the complex in soln. is very similar to the structure in the crystal. The structure presents a picture of how tandem G-U base pairs bind and position metal ions within the RNA major groove. The binding site is performed in the absence of metal ions, and presents a neg. pocket in the major groove with a variety of hydrogen-bond acceptors. Because G-U base pairs are such a common motif in RNA sequences, it is possible that this RNA-metal ion interaction is crit. in forming large complex RNA structures such as those found in the ribosome and self-splicing introns. This structure was detd. using cobalt (III) hexammine as an analog for hexahydrated magnesium, a technique that may be applicable to other RNA sequences. Metal hexammines may prove to be useful general probes for locating RNA metal ion binding sites in soln.
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    Wang, W. M., Zhao, J. W., Han, Q. W., Wang, G., Yang, G. C., Shallop, A. J., Liu, J., Gaffney, B. L., and Jones, R. A. (2009) Modulation of RNA metal binding by flanking bases: N-15 NMR evaluation of GC, tandem GU, and tandem GA sites Nucleosides Nucleotides Nucleic Acids 28, 424– 434
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    Modulation of RNA metal binding by flanking bases: 15N NMR evaluation of GC, tandem GU, and tandem GA sites
    Wang, Weimin; Zhao, Jianwei; Han, Qianwei; Wang, Gang; Yang, Gengcheng; Shallop, Anthony J.; Liu, Jane; Gaffney, Barbara L.; Jones, Roger A.
    Nucleosides, Nucleotides & Nucleic Acids (2009), 28 (5-7), 424-434CODEN: NNNAFY; ISSN:1525-7770. (Taylor & Francis, Inc.)
    15N NMR chem. shift changes in the presence of Mg(H2O)62+, Zn2+, Cd2+, and Co(NH3)63+ were used to probe the effect of flanking bases on metal binding sites in three different RNA motifs. We found that: for GC pairs, the presence of a flanking purine creates a site for the soft metals Zn2+ and Cd2+ only; a GG-UU motif selectively binds only Co(NH3)63+, while a UG-GU motif binds none of these metals; a 3' guanosine flanking the adenosine of a sheared GA·AG pair creates an unusually strong binding site that precludes binding to the cross-strand stacked guanosines within the tandem pair.
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    Colmenarejo, G. and Tinoco, I., Jr. (1999) Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme J. Mol. Biol. 290, 119– 135
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    Structure and Thermodynamics of Metal Binding in the P5 Helix of a Group I Intron Ribozyme
    Colmenarejo, Gonzalo; Tinoco, Ignacio, Jr.
    Journal of Molecular Biology (1999), 290 (1), 119-135CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
    The soln. structure of an RNA hairpin modeling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been detd. by NMR. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and pos. intermol. nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+mol. is in the major groove of tandem G·U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G·U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Neg. and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrns. of P5 show specific broadening of protons of the G·U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding consts. for the interaction of Co(NH3)63+and of Mg2+ to P5 were detd. by monitoring imino proton chem. shifts during titrn. of the RNA with the metal ions. Dissocn. consts. are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G·U base-pairs. The affinities of Co(NH3)63+and Mg2+ for the tandem G·U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G·C or A·U for G·U pairs also affected the binding, as expected. These structural and thermodn. results provide systematic new information about major groove metal ion binding in RNA. (c) 1999 Academic Press.
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    Gautheret, D., Konings, D., and Gutell, R. R. (1995) G·U base pairing motifs in ribosomal RNA RNA 1, 807– 814
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    G·U base pairing motifs in ribosomal RNA
    Gautheret, Daniel; Konings, Danielle; Gutell, Robin R.
    RNA (1995), 1 (8), 807-14CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    An increasing no. of recognition mechanisms in RNA are found to involve G.·U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G·U pairs. Approx. 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G·U pairs are consistent with previous proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G·U pairs in orientations that could cause severe stacking problems. In addn., three motifs displaying a conserved G·U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3'3'GU5', as well as G·U pairs flanked by a bulge loop 3' of U. The possible structures and functions of these recurrent motifs are discussed.
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    Gray, D. M. (1997) Derivation of nearest-neighbor properties from data on nucleic acid oligomers. 1. Simple sets of independent sequences and the influence of absent nearest neighbors Biopolymers 42, 783– 793
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    Derivation of nearest-neighbor properties from data on nucleic acid oligomers. I. Simple sets of independent sequences and the influence of absent nearest neighbors
    Gray, Donald M.
    Biopolymers (1997), 42 (7), 783-793CODEN: BIPMAA; ISSN:0006-3525. (John Wiley & Sons, Inc.)
    The constraints on combinations of nearest neighbors in nucleic acid sequences and the nos. of independent sequences needed to describe nearest-neighbor properties of oligomers and polymers are derived and summarized. It has been pointed out in previous work [D. M. Gray and I. Tinoco, Jr. (1970) Biopolymers, Vol. 9, pp. 223-244; R. F. Goldstein and A. S. Benight (1992) Biopolymers, Vol. 32, pp. 1679-1693] that these constraints restrict the information available from measurements of properties of sequence combinations. The emphasis in this paper is on the properties of oligomer sequences that vary in length, where each nucleotide or base pair at the end of the sequence makes a significant contribution to the measured property by interacting with its boundary of fixed sequence or solvent. In such cases it is not possible to det. values of properties of individual nearest neighbors, except for the like neighbors [e.g., d(A-A), d(G-G), d(T-T), and d(C-C) nucleotide neighbors in single-stranded DNA or d(A-A)/d(T-T) and d(G-G)/d(C-C) base pair neighbors in double-stranded DNA], solely from measurements of properties of different sequences. Even values for properties of the like neighbors cannot be detd. from such oligomeric sequences if the sequences are all of the same length. Nearest-neighbor properties of oligomer sequences that vary in length can be summarized in terms of the values for independent sets of sequences that are nearest neighbors and monomers all with boundaries of the fixed sequence or solvent. Straightforward combinations of the values for the independent sequences will give the values of the property for any dependent sequence, without explicit knowledge of the individual nearest-neighbor values. These considerations have important consequences for the derivation of widely used thermodn. parameters, as discussed in the following paper.
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    Nguyen, M.-T. and Schroeder, S. J. (2010) Consecutive terminal GU pairs stabilize RNA helices Biochemistry 49, 10574– 10581
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    Consecutive Terminal GU Pairs Stabilize RNA Helices
    Nguyen, Mai-Thao; Schroeder, Susan J.
    Biochemistry (2010), 49 (49), 10574-10581CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Consecutive GU pairs at the ends of RNA helixes provide significant thermodn. stability between -1.0 and -3.8 kcal/mol at 37°, which is equiv. to approx. 2 orders of magnitude in the value of a binding const. The thermodn. stabilities of GU pairs depend on the sequence, stacking orientation, and position in the helix. In contrast to GU pairs in the middle of a helix that may be destabilizing, all consecutive terminal GU pairs contribute favorable thermodn. stability. This work presents measured thermodn. stabilities for 30 duplexes contg. two, three, or four consecutive GU pairs at the ends of RNA helixes and a model to predict the thermodn. stabilities of terminal GU pairs. Imino proton NMR spectra show that the terminal GU nucleotides form hydrogen-bonded pairs. Different orientations of terminal GU pairs can have different conformations with equiv. thermodn. stabilities. These new data and prediction model will help improve RNA secondary structure prediction, identification of miRNA target sequences with GU pairs, and efforts to understand the fundamental phys. forces directing RNA structure and energetics.
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    Serra, M. J., Smolter, P. E., and Westhof, E. (2004) Pronouced instability of tandem IU base pairs in RNA Nucleic Acids Res. 32, 1824– 1828
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    Pronounced instability of tandem IU base pairs in RNA
    Serra, Martin J.; Smolter, Patricia E.; Westhof, Eric
    Nucleic Acids Research (2004), 32 (5), 1824-1828CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Optical melting was used to det. the stabilities of three series of RNA oligomers contg. tandem XU base pairs, GGCXUGCC (5'XU3'), GGCUXGCC (5'UX3') and GGCXXGGC/CCGUUCCG (5'XX3'), where X is either A, G or I (inosine). The helixes contg. tandem AU base pairs were the most stable in the first two series (5'XU3' and 5'UX3'), with an av. melting temp. ∼11° higher than the helixes with tandem 5'GU3' base pairs and 25° higher than the helixes with tandem 5'IU3' base pairs. For the third series (5'XX3'), the helix contg. tandem GG is the most stable, with an av. melting temp. ∼2° higher than the helix with tandem AA base pairs and ∼24° higher than the helix with tandem II base pairs. The thermodn. stability of the oligomers with tandem IU base pairs was also investigated as a function of magnesium ion concn. As with normal A-U or G-U tandem duplexes, the data could best be interpreted as non-specific binding of magnesium ions to the inosine-contg. RNA oligonucleotides.
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    Fukada, H. and Takahashi, K. (1998) Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride Proteins 33, 159– 166
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    Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride
    Fukada, Harumi; Takahashi, Katsutada
    Proteins: Structure, Function, and Genetics (1998), 33 (2), 159-166CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)
    Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically detd. in 0.1 M potassium chloride at temps. ranging from 5 to 45°. The values of the dissocn. const. were also detd. by means of potentiometric titrn. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temp. The enthalpy changes for the second dissocn. of phosphate and glycerol 2-phosphate where divalent anion is formed on dissocn. were fitted with the second order function of temp. rather than the first order. Temp. dependence of buffer pH calcd. by using the enthalpy and heat capacity changes obtained was in good agreement with the temp. variation of the pH values actually measured in the temp. range between 0 and 50° for all the buffers studied. On the basis of the results obtained, a numeric table showing the temp. dependence of pK values for the 18 buffers is presented.
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    Smallcombe, S. H. (1993) Solvent suppression with symmetrically-shifted pulses J. Am. Chem. Soc. 115, 4776– 4785
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    Solvent suppression with symmetrically-shifted pulses
    Smallcombe, Stephen H.
    Journal of the American Chemical Society (1993), 115 (11), 4776-85CODEN: JACSAT; ISSN:0002-7863.
    Sym.-shifted shaped pulses are used to suppress large solvent resonances such as water while allowing the observation of protons exchanging with or barrier under the water resonance. Sym.-shifted pulses are a family of pulses that have been optimized for non-excitation of the water resonance and can be seen as having historical precedence in, and many of the properties of, long rectangular pulses and binomial sequences. Linear prediction software coupled with data shifting is used to eliminate the undesirable frequency-dependent phase and baseline problems normally assocd. with pulses or excitation schemes of this length. Zero-frequency solvent subtraction is also described. Results are illustrated for DNA and protein samples in 90% water.
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    Grzesiek, S. and Bax, A. (1993) The importance of not saturating H2O in protein NMR - application to sensitivity enhancement and NOE measurements J. Am. Chem. Soc. 115, 12593– 12594
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    The importance of not saturating water in protein NMR. Application to sensitivity enhancement and NOE measurements
    Grzesiek, Stephan; Bax, Ad
    Journal of the American Chemical Society (1993), 115 (26), 12593-4CODEN: JACSAT; ISSN:0002-7863.
    The longitudinal relaxation time, T1, of H2O is nearly four times longer than the av. T1 of protein protons. If the intense water signal is suppressed by means of purge pulses this results in a satd. state for the slowly relaxing H2O signal, which via cross relaxation attenuates the protein spectrum. It is demonstrated that by designing pulse sequences in such a way that the water remains close to its equil. value during the entire expt. a significant enhancement in sensitivity can be obtained, particularly for the resonances of rapidly exchanging amide protons. The new approach returns the water magnetization to the z axis at the start of every acquisition, thereby improving water suppression compared to techniques that scramble magnetization by presatn. or purging pulses. This approach is demonstrated for 1H-15N correlation of the backbone amides in a complex between calmodulin and a tightly binding 26-residue peptide fragment of skeletal muscle myosin light chain kinase. Avoiding satn. of the water resonance is also shown to be important in measuring NOE interactions between water and protein protons, and for quant. measurement of the heteronuclear 15N-(1H) NOE. This latter measurement indicates that calmodulin residues 75 through 80 in the calmodulin peptide complex are subject to substantial internal dynamics, whereas the amides of residues 75 through 82 are subject to fast hydrogen exchange.
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    Piotto, M., Saudek, V., and Sklenář, V. (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions J. Biomol. NMR 2, 661– 665
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    Piotto, Martial; Saudek, Valdimir; Sklenar, Vladimir
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    A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aq. solns. (WATERGATE, WATER suppression by GrAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM soln. of a double-stranded DNA fragment in 90% H2O at 5° is presented.
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    Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: A multidimensional spectral processing system based on UNIX pipes J. Biomol. NMR 6, 277– 293
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    NMRPipe: a multidimensional spectral processing system based on UNIX pipes
    Delaglio, Frank; Grzesiek, Stephan; Vuister, Geerten W.; Zhu, Guang; Pfeifer, John; Bax, Ad
    Journal of Biomolecular NMR (1995), 6 (3), 277-93CODEN: JBNME9; ISSN:0925-2738. (ESCOM)
    The NMRPipe system is a UNIX software environment of processing, graphics, and anal. tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
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    Investigation of the Structural Basis for Thermodynamic Stabilities of Tandem GU Mismatches: Solution Structure of (rGAGGUCUC)2 by Two-Dimensional NMR and Simulated Annealing
    McDowell, Jeffrey A.; Turner, Douglas H.
    Biochemistry (1996), 35 (45), 14077-14089CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The duplex (rGAGGUCUC)2 contains the motif 3'-CUGG-5'5'-GGUC-3', which is unusually stable compared with other sym. tandem GU mismatches and occurs in the P5 helix of the group I intron of Tetrahymena thermophila. The three-dimensional soln. structure of (rGAGGUCUC)2 was detd. using two-dimensional NMR and a simulated annealing protocol. The structure is remarkably similar to the A-DNA crystal structure of (dGGGGTCCC)2 [Kneale, G., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 186, 805-814] which contains the analogous motif 3'-CTGG-5'5'-GGTC-3'. Incorporation of the 3'-CUGG-5'5'-GGUC-3' motif has little effect on backbone torsion angles and helical parameters compared with std. A-form. The only significant departure from A-form is a slight overtwisting 5' of the G in the GU mismatch and a displacement of the mismatches toward the minor groove. Inspection of stacking patterns of this structure and comparison with sym. tandem GT mismatches in A-DNA oligonucleotides from crystal structure data suggest that electrostatics are important in detg. motif stability.
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    Progress in Nuclear Magnetic Resonance Spectroscopy (1996), 29 (1/2), 51-127CODEN: PNMRAT; ISSN:0079-6565. (Elsevier)
    A review with 268 refs. Topics discussed include: RNA synthesis and purifn.; NMR spectroscopy; spectral assignments; constraints for structure detn.; structure calcns.;.
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    Chaires, Jonathan B.
    Biophysical Chemistry (1997), 64 (1-3), 15-23CODEN: BICIAZ; ISSN:0301-4622. (Elsevier)
    Monte Carlo simulations were used to reveal a subtle, hidden contribution to van't Hoff enthalpy ests. from small heat capacity changes. In simulated van't Hoff plots of ln K vs. 1/T, small heat capacity changes (-200 ≤ ΔCp ≤ +200 cal mol-1 K-1), for which curvature could not be discerned within the noise of the data, were found to systematically bias the slope, leading to apparently erroneous enthalpy ests. Nonlinear least squares anal. of the simulated van't Hoff plots further revealed that it is difficult to ext. statistically reliable ΔCp values from data with even modest noise levels. Ests. of ΔCp and ΔHvH were found to be highly correlated, indicating an ill-posed nonlinear fitting problem. Nonlinear fits were found in many cases to be statistically no better than simpler linear fits. These simulations show, however, that if an independent calorimetric enthalpy est. is available, apparent discrepancies between that value and ests. derived from van't Hoff plots may be used to infer the existence of a small heat capacity change and its sign. By this procedure, apparent differences between van't Hoff and calorimetric enthalpy ests. may be interpreted and reconciled. An important conclusion from these studies is that such differences most likely result for statistical reasons, rather than from underlying phys. causes.
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    Oligonucleotides (2003), 13 (6), 515-537CODEN: OLIGAJ; ISSN:1545-4576. (Mary Ann Liebert, Inc.)
    A review. The Tm is defined as temp. of melting or, more accurately, as temp. of midtransition. This term is often used for nucleic acids (DNA and RNA, oligonucleotides, and polynucleotides). A thermal denaturation expt. dets. the stability of the secondary structure of a DNA or RNA and aids in the choice of the sequences for antisense oligomers or PCR primers. Beyond a simple numerical value (the Tm), a thermal denaturation expt., in which the folded fraction of a structure is plotted vs. temp., yields important thermodn. information. The authors present classic problems encountered during these expts. and attempt to demonstrate that a no. of useful pieces of information can be extd. from these exptl. curves.
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    Biopolymers (1998), 44 (3), 309-319CODEN: BIPMAA; ISSN:0006-3525. (John Wiley & Sons, Inc.)
    A review with 76 refs. The thermodn. of RNA secondary structure formation in small model systems provides a database for predicting RNA structure from sequence. Methods for making these measurements are reviewed with emphasis on optical methods and treatment of exptl. errors. Anal. of exptl. results in terms of simple nearest-neighbor models is presented. Some measured sequence dependences of non-Watson-Crick motifs are discussed.
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    Furtig Boris; Richter Christian; Wohnert Jens; Schwalbe Harald
    Chembiochem : a European journal of chemical biology (2003), 4 (10), 936-62 ISSN:1439-4227.
    NMR spectroscopy is a powerful tool for studying proteins and nucleic acids in solution. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules, proteins, ions, and small ligands can be obtained for RNA molecules up to 100 nucleotides. This review provides insight into the resonance assignment methods that are the first and crucial step of all NMR studies, into the determination of base-pair geometry, into the examination of local and global RNA conformation, and into the detection of interaction sites of RNA. Examples of NMR investigations of RNA are given by using several different RNA molecules to illustrate the information content obtainable by NMR spectroscopy and the applicability of NMR techniques to a wide range of biologically interesting RNA molecules.
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    Reid, B. R., McCollumn, L., Ribeiro, N. S., Abbate, J., and Hurd, R. E. (1979) Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid Biochemistry 18, 3996– 4005
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    Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid
    Reid, Brian R.; McCollumn, Lillian; Ribeiro, N. Susan; Abbate, Joseph; Hurd, Ralph E.
    Biochemistry (1979), 18 (18), 3996-4005CODEN: BICHAW; ISSN:0006-2960.
    The low-field H bond ring NH proton NMR spectra of several tRNAs related to yeast tRNAPhe were examd. Several resonances were sensitive to Mg2+ and temp., suggesting that they are derived from tertiary base pairs. These same resonances could not be attributed to cloverleaf base pairs, as shown by exptl. assignment and ring current shift calcn. of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals ≥6 tertiary base pairs involving ring NH hydrogen bonds, which are apparently responsible for the extra resonances obsd. in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs were obsd. at the same position in the spectrum.
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    Johnston, P. D. and Redfield, A. G. (1981) Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons Biochemistry 20, 1147– 1156
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    Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons
    Johnston, Paul D.; Redfield, Alfred G.
    Biochemistry (1981), 20 (5), 1147-56CODEN: BICHAW; ISSN:0006-2960.
    Results directed primarily toward spectral assignment and nuclear spin dynamics are described for yeast tRNAPhe in 0.1M NaCl, pH 7. Mg2+ titrns. were performed. Changes in the spectrum occur for Mg2+/tRNA ratios of ∼2 and >10. Difference spectroscopy between 43 and 29° in zero Mg2+ concn., together with prior identification of the GU4 acceptor stem base pair, indicates early acceptor melting and is used to identify acceptor resonances. Transport of spin energy (spin diffusion) is described in tRNA together with a summary of relevant expts. A survey of nuclear Overhauser effects (NOE) between imino and arom. and amino protons is concluded, together with some recent conclusions based on Me NOE and expts. with tRNAs deuterated at the purine C8 position. Assignment of the imino NMR spectrum on the basis of these and previous data is reviewed and discussed in detail. Preliminary distance ests. based on the NOE for AU and GU4 base pairs are in reasonable agreement with the expected distances.
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    Deigan, K. E., Li, T. W., Mathews, D. H., and Weeks, K. M. (2009) Accurate SHAPE-directed RNA structure determination Proc. Natl. Acad. Sci. U.S.A. 106, 97– 102
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    Deigan, Katherine E.; Li, Tian W.; Mathews, David H.; Weeks, Kevin M.
    Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (1), 97-102CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Almost all RNAs can fold to form extensive base-paired secondary structures. Many of these structures then modulate numerous fundamental elements of gene expression. Deducing these structure-function relationships requires that it be possible to predict RNA secondary structures accurately. However, RNA secondary structure prediction for large RNAs, such that a single predicted structure for a single sequence reliably represents the correct structure, has remained an unsolved problem. Here, we demonstrate that quant., nucleotide-resoln. information from a SHAPE expt. can be interpreted as a pseudo-free energy change term and used to det. RNA secondary structure with high accuracy. Free energy minimization, by using SHAPE pseudofree energies, in conjunction with nearest neighbor parameters, predicts the secondary structure of deproteinized Escherichia coli 16S rRNA (>1300 nt) and a set of smaller RNAs (75-155 nt) with accuracies of up to 96-100%, which are comparable to the best accuracies achievable by comparative sequence anal.
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    Hart, J. M., Kennedy, S. D., Mathews, D. H., and Turner, D. H. (2008) NMR-assisted prediction of RNA secondary structure: Identification of a probable pseudoknot in the coding region of an R2 Retrotransposon J. Am. Chem. Soc. 130, 10233– 10239
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    NMR-Assisted Prediction of RNA Secondary Structure: Identification of a Probable Pseudoknot in the Coding Region of an R2 Retrotransposon
    Hart, James M.; Kennedy, Scott D.; Mathews, David H.; Turner, Douglas H.
    Journal of the American Chemical Society (2008), 130 (31), 10233-10239CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    As the rate of functional RNA sequence discovery escalates, high-throughput techniques for reliable structural detn. are becoming crucial for revealing the essential features of these RNAs in a timely fashion. Computational predictions of RNA secondary structure quickly generate reasonable models but suffer from several approxns., including overly simplified models and incomplete knowledge of significant interactions. Similar problems limit the accuracy of predictions for other self-folding polymers, including DNA and peptide nucleic acid (PNA). The work presented here demonstrates that incorporating unassigned data from simple NMR expts. into a dynamic folding algorithm greatly reduces the potential folding space of a given RNA and therefore increases the confidence and accuracy of modeling. This procedure has been packaged into an NMR-assisted prediction of secondary structure (NAPSS) algorithm that can produce pseudoknotted as well as non-pseudoknotted secondary structures. The method reveals a probable pseudoknot in the part of the coding region of the R2 retrotransposon from Bombyx mori that orchestrates second-strand DNA cleavage during insertion into the genome.
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    Batey, R. T., Rambo, R. P., and Doudna, J. A. (1999) Tertiary motifs in RNA structure and folding Angew. Chem., Int. Ed. 38, 2327– 2343
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    Batey, Robert T.; Rambo, Robert P.; Doudna, Jennifer A.
    Angewandte Chemie, International Edition (1999), 38 (16), 2327-2343CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
    A review with 140 refs. RNA plays a crit. role in mediating every step of the cellular information transfer pathway from DNA-encoded genes to functional proteins. Its diversity of biol. functions stems from the ability of RNA to act as a carrier of genetic information and to adopt complex 3-dimensional folds that create sites for chem. catalysis. At.-resoln. structures of several large RNA mols., detd. by x-ray crystallog., have elucidated some of the means by which a global fold is achieved. Within these RNAs are tertiary structural motifs that enable the highly anionic double-stranded helixes to tightly pack together to create a globular architecture. Here, an overview of the structures of these motifs and their contribution to the organization of large, biol. active RNAs is presented. Base stacking, participation of the ribose 2'-OH groups in H-bonding interactions, binding of divalent metal cations, noncanonical base pairing, and backbone topol. all serve to stabilize the global structure of RNA and play crit. roles in guiding the folding process. Studies of the RNA-folding problem, which is conceptually analogous to the protein-folding problem, have demonstrated that folding primarily proceeds through a hierarchical pathway in which domains assemble sequentially. Formation of the proper tertiary interactions between these domains leads to discrete intermediates along this pathway. Advances in the understanding of RNA structure have facilitated improvements in the techniques that are utilized in modeling the global architecture of biol. interesting RNAs that have been resistant to at.-resoln. structural anal.
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    Varani, G. and McClain, W. H. (2000) The G·U wobble base pair: a fundamental building block of RNA structure crucial to RNA function in diverse biological systems EMBO Rep. 1, 18– 23
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    The G·U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems
    Varani, Gabriele; McClain, William H.
    EMBO Reports (2000), 1 (1), 18-23CODEN: ERMEAX; ISSN:1469-221X. (Oxford University Press)
    A review with 47 refs. The G·U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodn. stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G·U or A·U base pairs. The G·U wobble base pair also has unique chem., structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites contg. G·U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biol. processes.
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    Allawi, H. T. and SantaLucia, J. (1997) Thermodynamics and NMR of internal G·T mismatches in DNA Biochemistry 36, 10581– 10594
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    Thermodynamics and NMR of Internal G·T Mismatches in DNA
    Allawi, Hatim T.; SantaLucia, John, Jr.
    Biochemistry (1997), 36 (34), 10581-10594CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Thermodn. of 39 oligonucleotides with internal G·T mismatches dissolved in 1 M NaCl were detd. from UV absorbance vs. temp. profiles. These data were combined with literature values of six sequences to derive parameters for 10 linearly independent trimer and tetramer sequences with G·T mismatches and Watson-Crick base pairs. The G·T mismatch parameters predict ΔG°37, ΔH°, ΔS°, and TM with av. deviations of 5.1%, 7.5%, 8.0%, and 1.4°, resp. These predictions are within the limits of what can be expected for a nearest-neighbor model. The data show that the contribution of a single G·T mismatch to helix stability is context dependent and ranges from +1.05 kcal/mol for AGA/TTT to -1.05 kcal/mol for CGC/GTG. Several tests of the applicability of the nearest-neighbor model to G·T mismatches are described. Anal. of imino proton chem. shifts show that structural perturbations from the G·T mismatches are highly localized. One-dimensional NOE difference spectra demonstrate that G·T mismatches form stable hydrogen-bonded wobble pairs in diverse contexts. Refined nearest-neighbor parameters for Watson-Crick base pairs are also presented.
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    Cate, Jamie H.; Doudna, Jennifer A.
    Structure (London) (1996), 4 (10), 1221-1229CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
    Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific 3-dimensional structures that confer activity. Here, the 2.8 Å crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 domain, provides the 1st detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold were examd. in detail. Heavy-atom derivs. used in the structure detn. revealed characteristics of some of the metal-binding sites in the P4-P6 domain. Although long-range RNA-RNA contacts within the mol. primarily involved the minor groove, Os hexaammine bound at 3 locations in the major groove. All 3 sites involved guanine and uracil nucleotides exclusively; 2 were formed by G·U wobble base pairs. In the native RNA, 2 of the sites were occupied by fully-hydrated Mg2+ ions. Sm3+ bound specifically to the RNA by displacing a Mg2+ in a region crit. to the folding of the entire domain. Bound at specific sites in the P4-P6 domain RNA, Os(III) hexaammine produced the high-quality heavy-atom deriv. used for structure detn. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivs. with hexaammine compds. The features of the obsd. metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G·U base pairs in rRNAs are Mg2+-binding sites.
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  76. 76
    Konforti, B. B., Abramovitz, D. L., Duarte, C. M., Karpeisky, A., Beigelman, L., and Pyle, A. M. (1998) Ribozyme catalysis from the major groove of group II intron domain 5 Mol. Cell 1, 433– 441
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    76
    Ribozyme catalysis from the major groove of group II intron domain 5
    Konforti, Boyana B.; Abramovitz, Dana L.; Duarte, Carlos M.; Karpeisky, Alex; Beigelman, Leonid; Pyle, Anna Marie
    Molecular Cell (1998), 1 (3), 433-441CODEN: MOCEFL ISSN:. (Cell Press)
    The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examd. We obsd. that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the crit. G (specifically N7 or O6) is essential for chem. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the crit. G could engage in crit. tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.
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  77. 77
    Adams, P. L., Stahley, M. R., Kosek, A. B., Wang, J., and Strobel, S. A. (2004) Crystal structure of a self-splicing group I intron with both exons Nature 430, 45– 50
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    77
    Crystal structure of a self-splicing group I intron with both exons
    Adams, Peter L.; Stahley, Mary R.; Kosek, Anne B.; Wang, Jimin; Strobel, Scott A.
    Nature (London, United Kingdom) (2004), 430 (6995), 45-50CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here the authors report the X-ray crystal structure (3.1-Å resoln.) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asym. positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.
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  78. 78
    Forconi, M., Sengupta, R. N., Piccirilli, J. A., and Herschlag, D. (2010) A rearrangement of the guanosine-binding site establishes an extended network of functional interactions in the tetrahymena group I ribozyme active site Biochemistry 49, 2753– 2762
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    78
    A Rearrangement of the Guanosine-Binding Site Establishes an Extended Network of Functional Interactions in the Tetrahymena Group I Ribozyme Active Site
    Forconi, Marcello; Sengupta, Raghuvir N.; Piccirilli, Joseph A.; Herschlag, Daniel
    Biochemistry (2010), 49 (12), 2753-2762CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Protein enzymes appear to use extensive packing and hydrogen bonding interactions to precisely position catalytic groups within active sites. Because of their inherent backbone flexibility and limited side chain repertoire, RNA enzymes face addnl. challenges relative to proteins in precisely positioning substrates and catalytic groups. Here, we use the group I ribozyme to probe the existence, establishment, and functional consequences of an extended network of interactions in an RNA active site. The group I ribozyme catalyzes a site-specific attack of guanosine on an oligonucleotide substrate. We previously detd. that the hydrogen bond between the exocyclic amino group of guanosine and the 2'-hydroxyl group at position A261 of the Tetrahymena group I ribozyme contributes to overall catalysis. We now use functional data, aided by double mutant cycles, to probe this hydrogen bond in the individual reaction steps of the catalytic cycle. Our results indicate that this hydrogen bond is not formed upon guanosine binding to the ribozyme but instead forms at a later stage of the catalytic cycle. Formation of this hydrogen bond is correlated with other structural rearrangements in the ribozyme's active site that are promoted by docking of the oligonucleotide substrate into the ribozyme's active site, and disruption of this interaction has deleterious consequences for the chem. transformation within the ternary complex. These results, combined with earlier results, provide insight into the nature of the multiple conformational steps used by the Tetrahymena group I ribozyme to achieve its active structure and reveal an intricate, extended network of interactions that is used to establish catalytic interactions within this RNA's active site.
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  79. 79
    Lipchock, S. V. and Strobel, S. A. (2008) A relaxed active site after exon ligation by the group I intron Proc. Natl. Acad. Sci. U.S.A 105, 5699– 5704
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    79
    A relaxed active site after exon ligation by the group I intron
    Lipchock, Sarah V.; Strobel, Scott A.
    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (15), 5699-5704CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two addnl. structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodn. measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.
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  80. 80
    Stahley, M. R., Adams, P. L., Wang, J., and Strobel, S. A. (2007) Structural metals in the group I intron: A ribozyme with a multiple metal ion core J. Mol. Biol. 372, 89– 102
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    80
    Structural Metals in the Group I Intron: A Ribozyme with a Multiple Metal Ion Core
    Stahley, Mary R.; Adams, Peter L.; Wang, Jimin; Strobel, Scott A.
    Journal of Molecular Biology (2007), 372 (1), 89-102CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We detd. the x-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addn. to 222 nucleotides of RNA, the model includes 18 Mg2+ and K+ ions. Five of the metals bind within 12 Å of the scissile phosphate and coordinate the majority of the oxygen atoms biochem. implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is crit. to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helixes. The group I intron has a rich history of biochem. efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochem. results to further understand the role of metal ions in group I intron structure and function.
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  81. 81
    Strobel, S. A. and Ortoleva-Donnelly, L. (1999) A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme Chem. Biol. 6, 153– 165
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    81
    A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme
    Strobel, Scott A.; Ortoleva-Donnelly, Lori
    Chemistry & Biology (1999), 6 (3), 153-165CODEN: CBOLE2; ISSN:1074-5521. (Current Biology Publications)
    The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chem. transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chem. basis of group I intron catalysis. Using nucleotide analog interference mapping and site-specific functional group substitution expts., we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chem. transition state by ∼ 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chem. step. These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.
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  82. 82
    Toor, N., Keating, K. S., Taylor, S. D., and Pyle, A. M. (2008) Crystal structure of a self-spliced group II intron Science 320, 77– 82
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    82
    Crystal Structure of a Self-Spliced Group II Intron
    Toor, Navtej; Keating, Kevin S.; Taylor, Sean D.; Pyle, Anna Marie
    Science (Washington, DC, United States) (2008), 320 (5872), 77-82CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Group II introns are self-splicing ribozymes that catalyze their own excision from precursor transcripts and insertion into new genetic locations. Here we report the crystal structure of an intact, self-spliced group II intron from Oceanobacillus iheyensis at 3.1 angstrom resoln. An extensive network of tertiary interactions facilitates the ordered packing of intron subdomains around a ribozyme core that includes catalytic domain V. The bulge of domain V adopts an unusual helical structure that is located adjacent to a major groove triple helix (catalytic triplex). The bulge and catalytic triplex jointly coordinate two divalent metal ions in a configuration that is consistent with a two-metal ion mechanism for catalysis. Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.
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  83. 83
    Xu, D., Landon, T., Greenbaum, N. L., and Fenley, M. O. (2007) The electrostatic characteristics of G·U wobble base pairs Nucleic Acids Res. 35, 3836– 3847
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    83
    The electrostatic characteristics of G•U wobble base pairs
    Xu, Darui; Landon, Theresa; Greenbaum, Nancy L.; Fenley, Marcia O.
    Nucleic Acids Research (2007), 35 (11), 3836-3847CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    G•U wobble base pairs are the most common and highly conserved non-Watson-Crick base pairs in RNA. Previous surface maps imply uniformly neg. electrostatic potential at the major groove of G•U wobble base pairs embedded in RNA helixes, suitable for entrapment of cationic ligands. In this work, we have used a Poisson-Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G•U wobble displays distinctly enhanced negativity compared with std. GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G•U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5kT/e, depending upon structural features. We propose that the negativity at the major groove of G•U wobble base pairs is detd. by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G•U sites in RNA are most likely to bind cationic ligands.
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  84. 84
    Chen, G., Znosko, B. M., Jiao, X. Q., and Turner, D. H. (2004) Factors affecting thermodynamic stabilities of RNA 3 × 3 internal loops Biochemistry 43, 12865– 12876
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    84
    Factors Affecting Thermodynamic Stabilities of RNA 3 × 3 Internal Loops
    Chen, Gang; Znosko, Brent M.; Jiao, Xiaoqi; Turner, Douglas H.
    Biochemistry (2004), 43 (40), 12865-12876CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Internal loops in RNA are important for folding and function. The 3 × 3 nucleotide internal loops are the smallest size sym. loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodn. and structural insights acquired for 3 × 3 loops should improve approxns. for stabilities of 3 × 3 and larger internal loops. Most natural 3 × 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides contg. different 3 × 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodn. stabilities of 3 × 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of ∼1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 × 3 loops is derived by multiple linear regression.
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  85. 85
    Serra, M. J., Baird, J. D., Dale, T., Fey, B. L., Retatagos, K., and Westhof, E. (2002) Effects of magnesium ions on the stabilization of RNA oligomers of defined structures RNA 8, 307– 323
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    85
    Effects of magnesium ions on the stabilization of RNA oligomers of defined structures
    Serra, Martin J.; Baird, John D.; Dale, Taraka; Fey, Bridget L.; Retatagos, Kimberly; Westhof, Eric
    RNA (2002), 8 (3), 307-323CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    Optical melting was used to det. the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concn. The oligomers included helixes composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concn. on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temp. or free energy) was three times greater than that obsd. for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes obsd. upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes obsd. in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the obsd. cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calcd.
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  86. 86
    Walter, A. E., Wu, M., and Turner, D. H. (1994) The stability and structure of tandem GA mismatches in RNA depend on closing base-pairs Biochemistry 33, 11349– 11354
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    86
    The Stability and Structure of Tandem GA Mismatches in RNA Depend on Closing Base Pairs
    Walter, Amy E.; Wu, Ming; Turner, Douglas H.
    Biochemistry (1994), 33 (37), 11349-54CODEN: BICHAW; ISSN:0006-2960.
    UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal sequence loops in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chem. shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.
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  87. 87
    Freier, S. M., Burger, B. J., Alkema, D., Neilson, T., and Turner, D. H. (1983) Effects of 3′ dangling end stacking on the stability of GGCC and CCGG double helixes Biochemistry 22, 6198– 6206
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    87
    Effects of 3' dangling end stacking on the stability of GGCC and CCGG double helixes
    Freier, Susan M.; Burger, Barbara J.; Alkema, Dirk; Neilson, Thomas; Turner, Douglas H.
    Biochemistry (1983), 22 (26), 6198-206CODEN: BICHAW; ISSN:0006-2960.
    The thermodn. properties for helix formation of 2 core mols., GGCC and CCGG, and pentanucleotides contg. either core plus a 3'-dangling nucleotide were measured spectrophotometrically. In 1M Na+, the order of stability was: GGCC ≈ GGCCp < pGGCC < GGCCC ≈ GGCCCp < GGCCUp < GGCCAp ≈ GGCCGp ≈ GGCCεAp (where εA is 1,N6-ethenoadenosine); CCGG < pCCGG < CCGGCp < CCGGUp < CCGGAp ≈ CCGGGp. In 0.01M Na+, the order of stability for the GGCC family did not change except that GGCC was more stable than the tetramers with a terminal phosphate. Thermodn. parameters obtained by using a 2-state model demonstrated that the stabilizing effect of a 3'-dangling end was enthalpic. The results indicated that stacking is an important contributor to nucleic acid stability. Sedimentation equil. expts. at 3° on GGCCGp in 1M Na+ and on GGCCAp in 0.01M Na+ indicated no aggregation of pentanucleotide helixes at strand concns. as high as 2 mM.
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  88. 88
    Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., and Turner, D. H. (1985) Contributions of dangling end stacking and terminal base-pair formation to the stabilities of XGGCCp, XCCGGp, XGGCCYp, and XCCGGYp helixes Biochemistry 24, 4533– 4539
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    88
    Contributions of dangling end stacking and terminal base-pair formation to the stabilities of XGGCCp, XCCGGp, XGGCCYp, and XCCGGYp helixes
    Freier, Susan M.; Alkema, Dirk; Sinclair, Alison; Neilson, Thomas; Turner, Douglas H.
    Biochemistry (1985), 24 (17), 4533-9CODEN: BICHAW; ISSN:0006-2960.
    The role of stacking in terminal base-pair formation was studied by comparison of the stability increments for dangling ends to those for fully formed base pairs. Thermodn. parameters were measured spectrophotometrically for helix formation of the hexanucleotides, AGGCCUp, UGGCCAp, CGGCCGp, GCCGGCp, and UCCGGAp, and for the corresponding pentanucleotides contg. a 5'-dangling end on the GGCCp or CCGGp core helix. In 1M NaCl at 1 × 10-4 M strands, a 5'-dangling nucleotide in this series increased the duplex melting temp. (Tm) only 0-4°, about the same as adding a 5'-phosphate. In contrast, as detd. previously, a 3'-dangling nucleotide increased the Tm at 1 × 10-4 M strands 7-23°, depending on the sequence. These results were consistent with stacking patterns obsd. in A-form RNA. The stability increments from terminal A-U, C-G, or U-A base pairs on GGCC or a terminal U-A pair on CCGG were nearly equal to the sums of the stability increments from the corresponding dangling ends. This suggested that stacking plays a large role in nucleic acid stability. The stability increment from the terminal base pairs in GCCGGCp, however, was ∼5-fold the sum of the corresponding dangling ends, suggesting H-bonding can also make important contributions.
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    Crick, F. H. C. (1966) Codon-anticodon pairing: the wobble hypothesis J. Mol. Biol. 19, 548– 555
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    89
    Codon-anticodon pairing: the wobble hypothesis
    Crick, F. H. C.
    Journal of Molecular Biology (1966), 19 (2), 548-55CODEN: JMOBAK; ISSN:0022-2836.
    It is suggested that while the standard base pairs may be used rather strictly in the 1st 2 positions of the triplet, there may be some wobble in the pairing of the third base. This hypothesis is explored systematically, and it is shown that such a wobble could explain the general nature of the degeneracy of the genetic code.
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  90. 90
    Chen, X. Y., McDowell, J. A., Kierzek, R., Krugh, T. R., and Turner, D. H. (2000) Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G·U pairs: One hydrogen bond for each G·U pair in r(GGCGUGCC)2 and two for each G·U pair in r(GAGUGCUC)2 Biochemistry 39, 8970– 8982
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    90
    Nuclear Magnetic Resonance Spectroscopy and Molecular Modeling Reveal That Different Hydrogen Bonding Patterns Are Possible for G·U Pairs: One Hydrogen Bond for Each G·U Pair in r(GGCGUGCC)2 and Two for Each G·U Pair in r(GAGUGCUC)2
    Chen, Xiaoying; McDowell, Jeffrey A.; Kierzek, Ryszard; Krugh, Thomas R.; Turner, Douglas H.
    Biochemistry (2000), 39 (30), 8970-8982CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    G·U pairs occur frequently and have many important biol. functions. The stability of sym. tandem G·U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G·U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G·U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochem. 34, 3204-3211]. In particular, at 37°, the motif 5'-CGUG-3' is less stable by approx. 3 kcal/mol compared with other sym. tandem G·U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The soln. structures of r(GAGUGCUC)2 and r(GGCGUGCC)2 duplexes have been detd. by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G·U pair region. The striking discovery is that in r(GGCGUGCC)2 each G·U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addn., the temp. dependence of the imino proton resonances is also consistent with the different no. of hydrogen bonds in the G·U pair. To test the NMR models, U or G in various G·U pairs were individually replaced by N3-methyluridine or isoguanosine, resp., thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.
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  91. 91
    Pan, Y. P., Priyakumar, U. D., and MacKerell, A. D. (2005) Conformational determinants of tandem GU mismatches in RNA: Insights from molecular dynamics simulations and quantum mechanical calculations Biochemistry 44, 1433– 1443
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    91
    Conformational Determinants of Tandem GU Mismatches in RNA: Insights from Molecular Dynamics Simulations and Quantum Mechanical Calculations
    Pan, Yongping; Priyakumar, U. Deva; MacKerell, Alexander D., Jr.
    Biochemistry (2005), 44 (5), 1433-1443CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Structure and energetic properties of base pair mismatches in duplex RNA have been the focus of numerous investigations due to their role in many important biol. functions. Such efforts have contributed to the development of models for secondary structure prediction of RNA, including the nearest-neighbor model. In RNA duplexes contg. GU mismatches, 5'-GU-3' tandem mismatches have a different thermodn. stability than 5'-UG-3' mismatches. In addn., 5'-GU-3' mismatches in some sequence contexts do not follow the nearest-neighbor model for stability. To characterize the underlying at. forces that det. the structural and thermodn. properties of GU tandem mismatches, mol. dynamics (MD) simulations were performed on a series of 5'-GU-3' and 5'-UG-3' duplexes in different sequence contexts. Overall, the MD-derived structural models agree well with exptl. data, including local deviations in base step helicoidal parameters in the region of the GU mismatches and the model where duplex stability is assocd. with the pattern of GU hydrogen bonding. Further anal. of the simulations, validated by data from quantum mech. calcns., suggests that the exptl. obsd. differences in thermodn. stability are dominated by GG interstrand followed by GU intrastrand base stacking interactions that dictate the one vs. two hydrogen bonding scenarios for the GU pairs. In addn., the inability of 5'-GU-3' mismatches in different sequence contexts to all fit into the nearest-neighbor model is indicated to be assocd. with interactions of the central four base pairs with the surrounding base pairs. The results emphasize the role of GG and GU stacking interactions on the structure and thermodn. of GU mismatches in RNA.
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  92. 92
    Biswas, R., Wahl, M. C., Ban, C., and Sundaralingam, M. (1997) Crystal structure of an alternating octamer r(GUAUGUA)dC with adjacent G·U wobble pairs J. Mol. Biol. 267, 1149– 1156
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    92
    Crystal structure of an alternating octamer r(GUAUGUA)dC with adjacent G·U wobble pairs
    Biswas, Roopa; Wahl, Markus C.; Ban, Changill; Sundaralingam, Muttaiya
    Journal of Molecular Biology (1997), 267 (5), 1149-1156CODEN: JMOBAK; ISSN:0022-2836. (Academic)
    The crystal structure of the RNA duplex, r(GUAUGUA)dC, with a 3'-terminal deoxy C residue, has been detd. at 1.38 Å resoln. The r(GUAUGU) hexameric consensus sequence is present at the exon-intron junction in pre-mRNAs of yeast and higher eukaryotic organisms. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 39.71 Å, c = 68.15 Å and γ = 120°C with one duplex in the asym. unit. The structure was solved using the mol. replacement method. The final model contains 332 atoms of the duplex and 67 solvent mols. The R-factor is 17.6% (Rfree of 23.1%) for 4035 reflections with F ≥ 1.5 σ(F) in the resoln. range 10.0 to 1.38 Å. The duplex is of the A-type with a pseudodyad relating the two strands. The RNA helix is slightly distorted, in spite of the presence of two adjacent G·U wobble base-pairs located at the center of the helix. The twist angle between the wobble pairs, 38.1°, is above the av. value and those between the wobble base-pairs and the flanking Watson-Crick base-pairs, 26.7° and 26.3°, resp., are lower than the av. values. The twist between the junction base-pairs are about 24°. The G·U wobble pairs are bridged by water mols. and solvated in the grooves. G·U base-pairs are as stable as the Watson-Crick A·U pairs and only slightly less stable than the G·C pairs accounting for their frequent occurrence in RNA.
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  93. 93
    Utsunomiya, R., Suto, K., Balasundaresan, D., Fukamizu, A., Kumar, P. K. R., and Mizuno, H. (2006) Structure of an RNA duplex r(GGCG(Br)UGCGCU)2 with terminal and internal tandem G·U base pairs Acta Crystallogr. D 62, 331– 338
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    93
    Structure of an RNA duplex r(GGCGBrUGCGCU)2 with terminal and internal tandem G.U base pairs
    Utsunomiya, Ryuji; Suto, Kyoko; Balasundaresan, Dhakshnamoorthy; Fukamizu, Akiyoshi; Kumar, Penmetcha K. R.; Mizuno, Hiroshi
    Acta Crystallographica, Section D: Biological Crystallography (2006), D62 (3), 331-338CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
    The crystal structure of a self-complementary RNA duplex r(GGCGBrUGCGCU)2 with terminal G.U and internal tandem G.U base pairs has been detd. at 2.1 Å resoln. The crystals belong to the tetragonal space group P43, with unit-cell parameters a = b = 37.69, c = 96.28 Å and two duplexes in the asym. unit. The two strands of each duplex are related by a pseudodyad axis. The structure was refined to final Rwork and Rfree values of 20.9 and 25.3%, resp. The duplexes stack in an end-to-end manner, forming infinite columns along the c axis. This is the first structural study of an RNA duplex contg. G.U pairs at the termini. The stacking overlaps of the terminal G.U base pairs with their adjacent Watson-Crick base pairs are larger than those of Watson-Crick base pairs of the 5'-YR-3'/3'-RY-5' type. The terminal G.U base pairs of neighboring duplexes are also stacked with each other. An alternating underwound-overwound pattern of the twist angles is seen at each step along the duplex. This observation is typical for internal tandem G.U pairs, while the terminal G.U base pairs exhibit high twist angles with the adjacent Watson-Crick pairs. The 3'-side of U of the internal G.U base pair, which is unstacked, appears to be stabilized by π-cation interaction with an Mg2+ ion.
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  94. 94
    Biswas, R. and Sundaralingam, M. (1997) Crystal structure of r(GUGUGUA)dC with tandem G·U/U·G wobble pairs with strand slippage J. Mol. Biol. 270, 511– 519
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    94
    Crystal structure of r(GUGUGUA)dC with tandem G·U/U·G wobble pairs with strand slippage
    Biswas, Roopa; Sundaralingam, Muttaiya
    Journal of Molecular Biology (1997), 270 (3), 511-519CODEN: JMOBAK; ISSN:0022-2836. (Academic)
    To better understand the frequent occurrence of adjacent wobble pairs in rRNAs we have detd. the crystal structure of the RNA duplex, r(GUGUGUA)dC with the 3'-terminal deoxy C residue. Two different crystal forms of the duplex were obtained and both belong to the rhombohedral space group, R3. Crystal form I has hexagonal unit cell dimensions, a = b = 40.82 Å and c = 66.09 Å and diffracts to 1.58 Å resoln., while crystal form II has a = b = 47.11 Å and c = 59.86 Å, diffracting only to 2.50 Å resoln. Both structures were solved by the mol. replacement method using different starting models. In spite of the large differences in the cell dimensions the overall structures in both crystals are similar. Instead of the expected blunt-end duplex with four consecutive G·U pairs, the slippage of the strands resulted in two different tandem G·U/U·G wobble pairs involving two of the central and two of the 5' overhang bases, still yielding a total of four wobble pairs. These tandem wobble pairs are flanked by two Watson-Crick pairs. The A-type duplexes stack in the familiar head-to-tail fashion forming a pseudocontinuous helix. The wobble pairs of the present motif II (G·U/U·G) structure stack with a low twist angle of 25.3° in contrast to that of motif I (U·G/G·U), 38.1°. The four wobble pairs are characteristically heavily hydrated in both the grooves accounting for their stability.
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  95. 95
    McDowell, J. A., He, L. Y., Chen, X. Y., and Turner, D. H. (1997) Investigation of the structural basis for thermodynamic stabilities of tandem GU wobble pairs: NMR structures of (rGGAGUUCC)2 and (rGGAUGUCC)2 Biochemistry 36, 8030– 8038
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    95
    Investigation of the Structural Basis for Thermodynamic Stabilities of Tandem GU Wobble Pairs: NMR Structures of (rGGAGUUCC)2 and (rGGAUGUCC)2
    McDowell, Jeffrey A.; He, Liyan; Chen, Xiaoying; Turner, Douglas H.
    Biochemistry (1997), 36 (26), 8030-8038CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The sym., tandem GU mismatch motifs, 3'-UGUA-5'5'-AUGU-3' and 3'-UUGA-5'5'-AGUU-3', which only differ in the mismatch order, have an av. difference in thermodn. stability of 2 kcal/mol at 37°. Thermodn. studies of duplexes contg. these motifs indicate the effect is largely localized to the mismatches and adjacent base pairs. The three-dimensional structures of two representative duplexes, (rGGAGUUCC)2 and (rGGAUGUCC)2, were detd. by two-dimensional NMR and a simulated annealing protocol. Local deviations are similar to other intrahelical GU mismatches with little effect on backbone torsion angles and a slight overtwisting between the base pair 5' of the G of the mismatch and the mismatch itself. Comparisons of the resulting stacking patterns along with electrostatic potential maps suggest that interactions between highly neg. electrostatic regions between base pairs may play a role in the obsd. thermodn. differences.
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    Masquida, B. and Westhof, E. (2000) On the wobble G·U and related pairs RNA 6, 9– 15
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    On the wobble GoU and related pairs
    Masquida, Benoit; Westhof, Eric
    RNA (2000), 6 (1), 9-15CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
    A review, with ∼60 refs. The wobble GoU pairs have been implicated in several biol. processes where RNA mols. play a key role. We review the geometrical and conformational properties of wobble GoU pairs on the basis of available crystal structures of RNAs at high resoln. The similarities with the wobble A+oC pairs and UoU pairs are illustrated, while the differences with the recently discovered bifurcated GU pairs are contrasted.
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  97. 97
    Jang, S. B., Hung, L. W., Jeong, M. S., Holbrook, E. L., Chen, X. Y., Turner, D. H., and Holbrook, S. R. (2006) The crystal structure at 1.5 Å resolution of an RNA octamer duplex containing tandem G·U basepairs Biophys. J. 90, 4530– 4537
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    97
    The crystal structure at 1.5 Å resolution of an RNA octamer duplex containing tandem G.U basepairs
    Jang, Se Bok; Hung, Li-Wei; Jeong, Mi Suk; Holbrook, Elizabeth L.; Chen, Xiaoying; Turner, Douglas H.; Holbrook, Stephen R.
    Biophysical Journal (2006), 90 (12), 4530-4537CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The crystal structure of the RNA octamer, 5'-GGCGUGCC-3' has been detd. from x-ray diffraction data to 1.5 Å resoln. In the crystal, this oligonucleotide forms five self-complementary double-helixes in the asym. unit. Tandem 5'GU/3'UG basepairs comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation among the five crystallog. duplexes and the soln. structure. The G.U pairs in the five duplexes of the crystal form two direct hydrogen bonds and are stabilized by water mols. that bridge between the base of guanine (N2) and the sugar (O2') of uracil. This contrasts with the NMR structure in which only one direct hydrogen bond is obsd. for the G.U pairs. The reduced stability of the r(CGUG)2 motif relative to the r(GGUC)2 motif may be explained by the lack of stacking of the uracil bases between the Watson-Crick and G.U pairs as obsd. in the crystal structure.
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  98. 98
    Deng, J. P. and Sundaralingam, M. (2000) Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G·G/U·U tandem wobble base pairs: comparison with other tandem G·U pairs Nucleic Acids Res. 28, 4376– 4381
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    98
    Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G.G/U.U tandem wobble base pairs: comparison with other tandem G.U pairs
    Deng, Junpeng; Sundaralingam, Muttaiya
    Nucleic Acids Research (2000), 28 (21), 4376-4381CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    We have detd. the crystal structure of the RNA octamer duplex r(guguuuac)/r(guaggcac) with a tandem wobble pair, G·G/U·U (motif III), to compare it with U·G/G·U (motif I) and G·U/U·G (motif II) and to better understand their relative stabilities. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 41.92 Å, c = 56.41 Å, and γ = 120°, with one duplex in the asym. unit. The structure was solved by the mol. replacement method at 1.9 Å resoln. and refined to a final R factor of 19.9% and Rfree of 23.3% for 2862 reflections in the resoln. range 10.0-1.9 Å with F ≥ 2σ(F). The final model contains 335 atoms for the RNA duplex and 30 water mols. The A-RNA stacks in the familiar head-to-tail fashion forming a pseudo-continuous helix. The uridine bases of the tandem U·G pairs have slipped towards the minor groove relative to the guanine bases and the uridine O2 atoms form bifurcated hydrogen bonds with the N1 and N2 of guanines. The N2 of guanine and O2 of uridine do not bridge the 'locked' water mol. in the minor groove, as in motifs I and II, but are bridged by water mols. in the major groove. A comparison of base stacking stabilities of motif III with motifs I and II confirms the result of thermodn. studies, motif I > motif III > motif II.
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  99. 99
    Shi, K., Wahl, M. C., and Sundaralingam, M. (1999) Crystal structure of an RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U base pairs Nucleic Acids Res. 27, 2196– 2201
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    99
    Crystal structure of an RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U base pairs
    Shi, Ke; Wahl, Markus; Sundaralingam, Muttaiya
    Nucleic Acids Research (1999), 27 (10), 2196-2201CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U and U·G wobble pairs sepd. by four Watson-Crick base pairs has been detd. to 2.5 A resoln. Crystals belong to the space group R3; a = 33.09 Å, α = 87.30 with a pseudodyad related duplex in the asym. unit. The structure was refined to a final Rwork of 17.5% and Rfree of 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3'-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3' penultimate phosphates are obsd. As expected, the wobble pairs are displaced with guanine toward the minor groove and uracil toward the major groove. The largest twist angles (37.70 and 40.57°) are at steps G1·C17/G2·U16 and U7·G11/C8·G10, while the smallest twist angles (28.24 and 27.27°) are at G2·U16/G3·C15 and C6·G12/U7·G11 and conform to the pseudo-dyad symmetry of the duplex. The mol. has two unequal kinks (17 and 11°) at the wobble sites and a third kink at the central G5 site which may be attributed to trans α(O5'-P), trans γ(C4'-C5') backbone conformations. The 2'-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water mols.
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  100. 100
    Alvarez-Salgado, F., Desvaux, H., and Boulard, Y. (2006) NMR assessment of the global shape of a non-labelled DNA dodecamer containing a tandem of G·T mismatches Magn. Reson. Chem. 44, 1081– 1089
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    100
    NMR assessment of the global shape of a non-labelled DNA dodecamer containing a tandem of G-T mismatches
    Alvarez-Salgado, Francisco; Desvaux, Herve; Boulard, Yves
    Magnetic Resonance in Chemistry (2006), 44 (12), 1081-1089CODEN: MRCHEG; ISSN:0749-1581. (John Wiley & Sons Ltd.)
    We have carried out a soln. study of two non-labeled self-complementary DNA dodecamers d(GACTGTACAGTC)2 and d(GACTGTGCAGTC)2 by NMR, the second sequence composed of two G-T mismatches. Structures were detd. using distances extd. from NOE effects alone or using both NOE and RDC constraints, measured in three different liq. cryst. media. We ensured that our data on the influence of the mesogen on the DNA structures, and the way in which the RDCs were incorporated as constraints in the protocol refinement, were consistent. We also tested the influence of different sets of RDCs and the best means of optimizing the calcn. of Da and R. Resoln. and accuracy of the ten best energy final structures were compared. The addn. of a small set of RDC constraints significantly improves the final detd. structures. We took advantage of the specificity of the RDC, i.e. it contains orientational information, and explored the global shape of the DNA duplexes; it was found that the duplexes do not have a large curvature. For the G-T base pair, we obsd., in this particular sequence (tandem of G-T mismatches), a new pattern of base pairing, which involved the formation of a bifurcated hydrogen bond.
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    Sugimoto, N., Kierzek, R., Freier, S. M., and Turner, D. H. (1986) Energetics of internal GU mismatches in ribooligonucleotide helixes Biochemistry 25, 5755– 5759
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    Energetics of internal GU mismatches in ribooligonucleotide helixes
    Sugimoto, Naoki; Kierzek, Ryszard; Freier, Susan M.; Turner, Douglas H.
    Biochemistry (1986), 25 (19), 5755-9CODEN: BICHAW; ISSN:0006-2960.
    Thermodn. parameters of helix formation were measured spectroscopically for 16 oligoribonucleotides contg. either internal GU mismatches or the corresponding AU pairs. Internal GU mismatches stabilize each helix, but not as much as the corresponding AU paris. The differences in the enthalpy and entropy changes of helix formation assocd. with replacing AU pairs with GU mismatches are less than previously realized. At both 25 and 37°, the decrease in helix stability assocd. with replacing an AU with a GU is also less than thought previously. Approxns. are suggested for predicting the effects of GU mismatches on helix stability.
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    Freier, S. M., Kierzek, R., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Free energy contributions of G·U and other terminal mismatches to helix stability Biochemistry 25, 3209– 3213
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    102
    Free energy contributions of G·U and other terminal mismatches to helix stability
    Freier, Susan M.; Kierzek, Ryszard; Caruthers, Marvin H.; Neilson, Thomas; Turner, Douglas H.
    Biochemistry (1986), 25 (11), 3209-13CODEN: BICHAW; ISSN:0006-2960.
    Thermodn. parameters of helix formation were measured spectroscopically for 7 hexaribonucleotides contg. a GC tetramer core and G·U or other terminal base-pair mismatches. The free energies of helix formation were compared with those for the tetramer core alone and with those for the hexamer with 6 Watson-Crick base pairs. In 1M NaCl at 37°, the free energy of a terminal G·U mismatch is about equal to that of the corresponding A·U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to the free energy (37°) for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G·U mismatches and G·C pairs suggest that when stacking is weak, the addnl. H bond in the G·C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.
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  103. 103
    Testa, S. M., Disney, M. D., Turner, D. H., and Kierzek, R. (1999) Thermodynamics of RNA-RNA duplexes with 2-or 4-thiouridines: Implications for antisense design and targeting a group I intron Biochemistry 38, 16655– 16662
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    103
    Thermodynamics of RNA-RNA Duplexes with 2- or 4-Thiouridines: Implications for Antisense Design and Targeting a Group I Intron
    Testa, Stephen M.; Disney, Matthew D.; Turner, Douglas H.; Kierzek, Ryszard
    Biochemistry (1999), 38 (50), 16655-16662CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Antisense compds. are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compd. and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s2U or s4U) show that s2U can increase the stability of base pairing with A more than with G, while s4U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s4U-G pair. To test the effects of s2U and s4U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs2U), and r(AUGACs4U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compds. targeting cellular RNA.
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  104. 104
    He, L., Kierzek, R., SantaLucia, J., Walter, A. E., and Turner, D. H. (1991) Nearest-neighbor parameters for G·U mismatches - 5′GU3′/3′UG5′ is destabilizing in the contexts CGUG/GUGC, UGUA/AUGU, and AGUU/UUGU but stabilizing in GGUC/CUGG Biochemistry 30, 11124– 11132
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    Biochemistry (1991), 30 (46), 11124-32CODEN: BICHAW; ISSN:0006-2960.
    Thermodn. parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides contg. G·U mismatches. The results are used to det. nearest-neighbor parameters for helix propagation by G·U mismatches. Surprisingly, the 5'GU3'/3'UG5' nearest-neighbor free energy increment is unfavorable in the contexts CGUG/GUGC'/UGUA/AUGU', and AGUU/UUGA but favorable in the context GGUC/CUGG'. This is a non-nearest-neighbor effect. In contrast, the 5'UG3'/3'GU5' free energy increment is favorable and independent of context. CD and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G·U mismatches have two slowly exchanging imino protons. The imino resonances for the G·U mismatches in GGAGU UCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temp. than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G·U mismatches in GGAUGUCC remain sharp at high temp. The improved parameters for G·U mismatches should improve predictions of RNA structure from sequence.
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    Thermodynamics of nonsymmetric tandem mismatches adjacent to G·C base pairs in RNA
    Xia, Tianbing; McDowell, Jeffrey A.; Turner, Douglas H.
    Biochemistry (1997), 36 (41), 12486-12497CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The thermodn. stabilities and structures of a series of RNA duplexes contg. nonsym. tandem mismatches in the context of 5'GAGXYGAG3'/3'CUCWZCUC5', where XY/WZ are tandem mismatches, were studied by UV melting and imino proton NMR. The contribution of one mismatch to the free energy increment for tandem mismatch formation depends on the identity of the other mismatch. Imino proton NMR indicates that this is partly because the structure of a mismatch is dependent on the adjacent mismatch. The results suggest that differences in size, shape, and hydrogen bonding of the adjacent mismatches play important roles in detg. loop stability. A model for predicting stabilities of all possible tandem mismatches is proposed based on these and previous results.
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    Sequence dependence for the energetics of terminal mismatches in ribooligonucleotides
    Sugimoto, Naoki; Kierzek, Ryszard; Turner, Douglas H.
    Biochemistry (1987), 26 (14), 4559-62CODEN: BICHAW; ISSN:0006-2960.
    Stability increments of terminal mismatches on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 15 oligoribonucleotides contg. the core sequences and various mismatches. Free energy increments for mismatches in this series range from -0.5 to -1.1 kcal/mol. These increments for mismatches on AU base pairs are smaller than those measured previously on GC base pairs. The terminal GU mismatches in the sequences GAUGCAUUp and UAUGCAUGp add approx. the same stability increment as the corresponding terminal AU mismatch. The stability increments for pyrimidine-pyrimidine and pyrimidine-purine mismatches can be approximated within -0.3 kcal/mol by adding the stability increments for the corresponding 3' and 5' unpaired nucleotides (dangling ends). Stability increments for purine-mismatches are approximated well by the stability increment for the corresponding 3' dangling end made more favorable by 0.2 kcal/mol. These approxns. are used to provide a table of stability increments for all 48 possible sequences of mismatches.
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    The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides
    Ziomek, Krzysztof; Kierzek, Elzbieta; Biala, Ewa; Kierzek, Ryszard
    Biophysical Chemistry (2002), 97 (2-3), 233-241CODEN: BICIAZ; ISSN:0301-4622. (Elsevier Science B.V.)
    The presence of various modifications within oligomers changes their thermodn. stability. To get more systematic data, we measured effects of 5- and 6-substituted uridine on thermal stability of (AUCUMod.AGAU)2 and (AUCUAGAUMod.)2. Collected results lead to the following conclusions: (i) 5-halogenated and 5-alkylated substituents of the uridine affect thermal stability of the RNA duplexes differently. Moreover, the 5-fluorouridine changes stability of the RNA duplexes opposite to remaining 5-halogenouridines; (ii) for oligomers contg. 5-chloro, 5-bromo or 5-iodouridine stronger hydrogen bond formed between oxygen-4 of the 5-halogenated uracil and 6-amino group of the adenine is presumably responsible for stabilizing effect; (iii) placing of A-U5R base pairs closer to the end of the duplex enhance thermal stability relatively to oligomer with central position of this base pair; (iv) the effects of 5-substituents are additive, particularly for substituents which stabilize RNA duplexes; (v) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine present at internal position of A-UMod. inhibit duplexes formation; (vi) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine placed as terminal base pairs stabilize the duplexes mostly via 3'-dangling end effect.
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    Thermodynamic Stabilities of Internal Loops with GU Closing Pairs in RNA
    Schroeder, Susan J.; Turner, Douglas H.
    Biochemistry (2001), 40 (38), 11509-11517CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodn. parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes contg. small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodn. stabilities of asym. internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for sym. internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.
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    Factors Affecting the Thermodynamic Stability of Small Asymmetric Internal Loops in RNA
    Schroeder, Susan J.; Turner, Douglas H.
    Biochemistry (2000), 39 (31), 9257-9274CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Optical melting expts. were used to det. the thermodn. parameters for oligoribonucleotides contg. small asym. internal loops. The results show a broad range of thermodn. stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR expts. provide evidence for possible hydrogen bonding in GA and UU mismatches in some asym. loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodn. stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 × 3 and 1 × 3 internal loops.
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    Freier, S. M., Sinclair, A., Neilson, T., and Turner, D. H. (1985) Improved free energies for G·C base-pairs J. Mol. Biol. 185, 645– 647
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    Improved free energies for G·C base-pairs
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    Thermodn. parameters of helix formation are reported for 7 oligoribonucleotides contg. only G·C pairs. These data are used with the nearest-neighbor model to calc. enthalpies and free energies of base pair formation for G·C pairs. For helix initiation, the free energy change at 37°, ΔG037, is +3.9 kcal/mol; for helix propagation, the ΔG037 values are -2.3, -3.2, and -3.3 kcal/mol for C-G, G-G, and G-C neighbors, resp.
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    Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Improved free-energy parameters for predictions of RNA duplex stability Proc. Natl. Acad. Sci. U.S.A. 83, 9373– 9377
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    Improved free-energy parameters for predictions of RNA duplex stability
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    Proceedings of the National Academy of Sciences of the United States of America (1986), 83 (24), 9373-7CODEN: PNASA6; ISSN:0027-8424.
    Thermodn. parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize exptl. errors in ΔG° at 37°. These parameters predict melting temps. of most oligonucleotide duplexes within 5°. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G·U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.
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    Freier, S. M., Sugimoto, N., Sinclair, A., Alkema, D., Neilson, T., Kierzek, R., Caruthers, M. H., and Turner, D. H. (1986) Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids Biochemistry 25, 3214– 3219
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    Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids
    Freier, Susan M.; Sugimoto, Naoki; Sinclair, Alison; Alkema, Dirk; Neilson, Thomas; Kierzek, Ryszard; Caruthers, Marvin H.; Turner, Douglas H.
    Biochemistry (1986), 25 (11), 3214-19CODEN: BICHAW; ISSN:0006-2960.
    The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers, XGCGCp and GCGCYp, contg. the GCGC core plus a dangling end (X and Y are various nucleosides). Each 5' dangling end increases helix stability at 37° ∼0.2 kcal/mol and each 3' end increases helix stability 0.8-1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported previously for the GGCC core, indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G·C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of stacking and pairing. If pairing contributions are due to H bonding, the results suggest stacking and H bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment assocd. with forming a H bond is estd. to be -1 kcal/mol H bond.
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    Sugimoto, N., Kierzek, R., and Turner, D. H. (1987) Sequence dependence for the energetics of dangling ends and terminal base pairs in ribooligonucleotides Biochemistry 26, 4554– 4558
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    113
    Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid
    Sugimoto, Naoki; Kierzek, Ryszard; Turner, Douglas H.
    Biochemistry (1987), 26 (14), 4554-8CODEN: BICHAW; ISSN:0006-2960.
    Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides contg. the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the addnl. stability from a 3' dangling end on an AU base pair is less than that on a GC base pair. The sequence dependence provides a test for the importance of dangling ends for various RNa interactions. Correlations are suggested with codon context effects and with the 3-dimensional structure of yeast phenylalanine tRNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is assocd. with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA mols. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment assocd. with forming a H bond can be about -1 kcal/mol of H bond.
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    Burkard, M. E. and Turner, D. H. (2000) NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs Biochemistry 39, 11748– 11762
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    NMR Structures of r(GCAGGCGUGC)2 and Determinants of Stability for Single Guanosine-Guanosine Base Pairs
    Burkard, Mark E.; Turner, Douglas H.
    Biochemistry (2000), 39 (38), 11748-11762CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')2, in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, resp. An addnl. hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-Rp phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')2, the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodn. data for a variety of duplexes contg. pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')2, suggesting similar GG conformations.
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    Petersheim, M. and Turner, D. H. (1983) Base-stacking and base-pairing contributions to helix stability: thermodynamics of double-helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp Biochemistry 22, 256– 263
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    Base-stacking and base-pairing contributions to helix stability: thermodynamics of double-helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp
    Petersheim, Matthew; Turner, Douglas H.
    Biochemistry (1983), 22 (2), 256-63CODEN: BICHAW; ISSN:0006-2960.
    The thermodn. of double-helix formation in 1M NaCl were measured spectrophotometrically for CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp. The results indicate that addnl. double-helical stability is conferred by the terminal unpaired bases. The 3'-adenine stabilizes the double-helix more than the 5'-adenine or the 3'-uracil. The increased stability is due to a more favorable enthalpy change for double-helix formation. Comparison of the thermodn. for CCGG, ACCGGp, CCGGUp, and ACCGGUp indicates that stacking interactions are somewhat more important than pairing interactions in detg. the stability of the terminal AU base pairs in ACCGGUp.
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    Kierzek, R., Caruthers, M. H., Longfellow, C. E., Swinton, D., Turner, D. H., and Freier, S. M. (1986) Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability Biochemistry 25, 7840– 7846
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    Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability
    Kierzek, Ryszard; Caruthers, Marvin H.; Longfellow, Carl E.; Swinton, David; Turner, Douglas H.; Freier, Susan M.
    Biochemistry (1986), 25 (24), 7840-6CODEN: BICHAW; ISSN:0006-2960.
    Solid-phase method using a phosphoramidite approach was used for synthesis of oligoribonucleotide pairs of oligomers with identical nearest neighbors but different sequences. Comparison of thermodn. parameters for these pairs provides a test of the nearest-neighbor hypothesis for predication of helix stability. In general, pairs of sequences with identical nearest neighbors have enthalpy and entropy changes for helix formation that differ by 8% on av., ΔG°37 that differ by 6% on av., and melding temps. within 0-5° of each other. These limits are typical of the accuracy that should be expected from nearest-neighbor predictions of RNA helix stability. UCAUGA and UGAUCA have the nearest neighbors but melting temps. that differ by 7°. This suggests some sequences will not be approximated well by the nearest-neighbor model.
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    Kierzek, R., Burkard, M. E., and Turner, D. H. (1999) Thermodynamics of single mismatches in RNA duplexes Biochemistry 38, 14214– 14223
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    Thermodynamics of Single Mismatches in RNA Duplexes
    Kierzek, Ryszard; Burkard, Mark E.; Turner, Douglas H.
    Biochemistry (1999), 38 (43), 14214-14223CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The thermodn. properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR expts. The free energy increments at 37 °C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approxns. are suggested for predicting stabilities of single mismatches in short RNA duplexes.
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  • Abstract

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    Figure 1

    Figure 1. 1D 1H imino spectra of r(CCGAAUUUGG)2 from 0 to 45 °C.

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    Figure 2

    Figure 2. 1D imino NMR spectra for some RNA duplexes with GU pairs. Spectra were acquired at 0 °C for r(AGGCUU)2 and r(AUGCGU)2; 1 °C for r(AGUCGAUU)2; 5 °C for others.

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  • References

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      Thermodynamic Parameters for an Expanded Nearest-Neighbor Model for Formation of RNA Duplexes with Watson-Crick Base Pairs
      Xia, Tianbing; SantaLucia, John, Jr.; Burkard, Mark E.; Kierzek, Ryszard; Schroeder, Susan J.; Jiao, Xiaoqi; Cox, Christopher; Turner, Douglas H.
      Biochemistry (1998), 37 (42), 14719-14735CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Improved thermodn. parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes contg. only Watson-Crick base pairs. To test end or base compn. effects, new sets of duplexes are included that have identical nearest neighbors, but different base compns. and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of ΔG°37 from a nearest-neighbor model. A phys. model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total no. of Watson-Crick hydrogen bonds on the base compn. of a duplex. On av., the new parameters predict ΔG°37, ΔH°, ΔS°, and TM within 3.2%, 6.0%, 6.8%, and 1.3°, resp. These predictions are within the limit of the model, based on exptl. results for duplexes predicted to have identical thermodn. parameters.
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    12. 12
      Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure J. Mol. Biol. 288, 911– 940
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      12
      Expanded Sequence Dependence of Thermodynamic Parameters Improves Prediction of RNA Secondary Structure
      Mathews, David H.; Sabina, Jeffrey; Zuker, Michael; Turner, Douglas H.
      Journal of Molecular Biology (1999), 288 (5), 911-940CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodn. parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent expts. Addnl. algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures detd. by comparative sequence anal. was assembled to allow optimization of parameters not based on expts. and to test the accuracy of the algorithm. On av., the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on av., has 86 % of known base-pairs. Exptl. constraints, derived from enzymic and FMN cleavage, improve the accuracy of structure predictions. (c) 1999 Academic Press.
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    13. 13
      Turner, D. H. (2000) Conformational changes, in Nucleic Acids: Structures, Properties, and Functions (Bloomfield, V. A., Crothers, D. M., and Tinoco, J., I., Eds.) pp 259– 334, University Science Books, Herndon, VA.
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      Washietl, S., Hofacker, I. L., and Stadler, P. F. (2005) Fast and reliable prediction of noncoding RNAs Proc. Natl. Acad. Sci. U.S.A. 102, 2454– 2459
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      14
      Fast and reliable prediction of noncoding RNAs
      Washietl, Stefan; Hofacker, Ivo L.; Stadler, Peter F.
      Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (7), 2454-2459CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The authors report an efficient method for detecting functional RNAs. The approach, which combines comparative sequence anal. and structure prediction, already has yielded excellent results for a small no. of aligned sequences and is suitable for large-scale genomic screens. It consists of two basic components: (i) a measure for RNA secondary structure conservation based on computing a consensus secondary structure, and (ii) a measure for thermodn. stability, which, in the spirit of a z score, is normalized with respect to both sequence length and base compn. but can be calcd. without sampling from shuffled sequences. Functional RNA secondary structures can be identified in multiple sequence alignments with high sensitivity and high specificity. The authors demonstrate that this approach is not only much more accurate than previous methods but also significantly faster. The method is implemented in the program RNAZ. The authors screened all alignments of length n ≥ 50 in the Comparative Regulatory Genomics database, which compiles conserved noncoding elements in upstream regions of orthologous genes from human, mouse, rat, Fugu, and zebrafish. The authors recovered all of the known noncoding RNAs and cis-acting elements with high significance and found compelling evidence for many other conserved RNA secondary structures not described so far to their knowledge.
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    15. 15
      Uzilov, A., Keegan, J., and Mathews, D. H. (2006) Detection of non-coding RNAs on the basis of predicted secondary structure formation free energy change BMC Bioinformatics 7, 173
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      15
      Detection of non-coding RNAs on the basis of predicted secondary structure formation free energy change
      Uzilov Andrew V; Keegan Joshua M; Mathews David H
      BMC bioinformatics (2006), 7 (), 173 ISSN:.
      BACKGROUND: Non-coding RNAs (ncRNAs) have a multitude of roles in the cell, many of which remain to be discovered. However, it is difficult to detect novel ncRNAs in biochemical screens. To advance biological knowledge, computational methods that can accurately detect ncRNAs in sequenced genomes are therefore desirable. The increasing number of genomic sequences provides a rich dataset for computational comparative sequence analysis and detection of novel ncRNAs. RESULTS: Here, Dynalign, a program for predicting secondary structures common to two RNA sequences on the basis of minimizing folding free energy change, is utilized as a computational ncRNA detection tool. The Dynalign-computed optimal total free energy change, which scores the structural alignment and the free energy change of folding into a common structure for two RNA sequences, is shown to be an effective measure for distinguishing ncRNA from randomized sequences. To make the classification as a ncRNA, the total free energy change of an input sequence pair can either be compared with the total free energy changes of a set of control sequence pairs, or be used in combination with sequence length and nucleotide frequencies as input to a classification support vector machine. The latter method is much faster, but slightly less sensitive at a given specificity. Additionally, the classification support vector machine method is shown to be sensitive and specific on genomic ncRNA screens of two different Escherichia coli and Salmonella typhi genome alignments, in which many ncRNAs are known. The Dynalign computational experiments are also compared with two other ncRNA detection programs, RNAz and QRNA. CONCLUSION: The Dynalign-based support vector machine method is more sensitive for known ncRNAs in the test genomic screens than RNAz and QRNA. Additionally, both Dynalign-based methods are more sensitive than RNAz and QRNA at low sequence pair identities. Dynalign can be used as a comparable or more accurate tool than RNAz or QRNA in genomic screens, especially for low-identity regions. Dynalign provides a method for discovering ncRNAs in sequenced genomes that other methods may not identify. Significant improvements in Dynalign runtime have also been achieved.
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    16. 16
      Gruber, A. R., Neuböck, R., Hofacker, I. L., and Washietl, S. (2007) The RNAz web server: prediction of thermodynamically stable and evolutionarily conserved RNA structures Nucleic Acids Res. 35, W335– W338
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      16
      The RNAz web server: prediction of thermodynamically stable and evolutionarily conserved RNA structures
      Gruber Andreas R; Neubock Richard; Hofacker Ivo L; Washietl Stefan
      Nucleic acids research (2007), 35 (Web Server issue), W335-8 ISSN:.
      Many non-coding RNA genes and cis-acting regulatory elements of mRNAs contain RNA secondary structures that are critical for their function. Such functional RNAs can be predicted on the basis of thermodynamic stability and evolutionary conservation. We present a web server that uses the RNAz algorithm to detect functional RNA structures in multiple alignments of nucleotide sequences. The server provides access to a complete and fully automatic analysis pipeline that allows not only to analyze single alignments in a variety of formats, but also to conduct complex screens of large genomic regions. Results are presented on a website that is illustrated by various structure representations and can be downloaded for local view. The web server is available at: rna.tbi.univie.ac.at/RNAz.
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      Reiche, K. and Stadler, P. F. (2007) RNAstrand: reading direction of structured RNAs in multiple sequence alignments Algorithm. Mol. Biol. 2, 6
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      17
      RNAstrand: reading direction of structured RNAs in multiple sequence alignments
      Reiche Kristin; Stadler Peter F
      Algorithms for molecular biology : AMB (2007), 2 (), 6 ISSN:.
      MOTIVATION: Genome-wide screens for structured ncRNA genes in mammals, urochordates, and nematodes have predicted thousands of putative ncRNA genes and other structured RNA motifs. A prerequisite for their functional annotation is to determine the reading direction with high precision. RESULTS: While folding energies of an RNA and its reverse complement are similar, the differences are sufficient at least in conjunction with substitution patterns to discriminate between structured RNAs and their complements. We present here a support vector machine that reliably classifies the reading direction of a structured RNA from a multiple sequence alignment and provides a considerable improvement in classification accuracy over previous approaches. SOFTWARE: RNAstrand is freely available as a stand-alone tool from http://www.bioinf.uni-leipzig.de/Software/RNAstrand and is also included in the latest release of RNAz, a part of the Vienna RNA Package.
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    18. 18
      White, S. A., Nilges, M., Huang, A., Brunger, A. T., and Moore, P. B. (1992) NMR analysis of helix-I from the 5S RNA of Escherichia coli Biochemistry 31, 1610– 1621
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      18
      NMR analysis of helix I from the 5S RNA of Escherichia coli
      White, S. A.; Nilges, M.; Huang, A.; Brunger, A. T.; Moore, P. B.
      Biochemistry (1992), 31 (6), 1610-21CODEN: BICHAW; ISSN:0006-2960.
      The structure of helix I of the 5 S rRNA from E. coli has been detd. using a nucleolytic digest fragment of the intact mol. The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5 S rRNA, contains a G-U (guanine-uracil) pair and has unpaired bases at its termini. Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling const. information have been used to calc. structural models for it using the full relaxation matrix algorithm of the mol. dynamics program XPLOR. Helix I has A-type helical geometry, as expected. Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair on its 3' side. This stacking pattern maximized interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G. These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe and A-form DNA. The configurations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5 S RNA, are less well-detd., and their sugar puckers are intermediate between C2'- and C3'-endo, on av.
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    19. 19
      Szymański, M., Barciszewska, M. Z., Erdmann, V. A., and Barciszewski, J. (2000) An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs Mol. Biol. Evol. 17, 1194– 1198
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      19
      An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs
      Szymanski, Maciej; Barciszewska, Miroslawa Z.; Erdmann, Volker A.; Barciszewski, Jan
      Molecular Biology and Evolution (2000), 17 (8), 1194-1198CODEN: MBEVEO; ISSN:0737-4038. (Society for Molecular Biology and Evolution)
      The structure-function relationship in RNA mols. is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an anal. of the distribution of G-U pairs in eukaryotic 5S rRNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.
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    20. 20
      Sprinzl, M. and Vassilenko, K. S. (2005) Compilation of tRNA sequences and sequences of tRNA genes Nucleic Acids Res. 33, D139– D140
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      20
      Compilation of tRNA sequences and sequences of tRNA genes
      Sprinzl, Mathias; Vassilenko, Konstantin S.
      Nucleic Acids Research (2005), 33 (Database), D139-D140CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Maintained at the Universitaet Bayreuth, Bayreuth, Germany, the Compilation of tRNA Sequences and Sequences of tRNA Genes is accessible at the URL http://www.tRNA.uni-bayreuth.de with mirror site located at the Institute of Protein Research, Pushchino, Russia (http://alpha.protres.ru/trnadbase). The compilation is a searchable, periodically updated database of currently available tRNA sequences. The present version of the database contains a new Genomic tRNA compilation including the sequences of tRNA genes from genomic sequences published up to July 2003. It consists of about 5800 tRNA gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya. The former Compilation of tRNA Genes (up to the end of 1998) and the updated compilation tRNA Sequences (561 entries) are also supported by the new software. The database can be explored by using multiple search criteria and sequence templates. The database provides a service that allows to obtain statistical information on the occurrences of certain bases at given positions of the tRNA sequences. This allows phylogenic studies and search for identity elements in respect to interactions of tRNAs with various enzymes.
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    21. 21
      Limmer, S., Reif, B., Ott, G., Arnold, L., and Sprinzl, M. (1996) NMR evidence for helix geometry modifications by a G-U wobble base pair in the acceptor arm of E-coli tRNA(Ala) FEBS Lett. 385, 15– 20
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      NMR evidence for helix geometry modifications by a G-U wobble base pair in the acceptor arm of E. coli tRNAAla
      Limmer, Stefan; Reif, Bernd; Ott, Geunther; Arnold, Lubos; Sprinzl, Mathias
      FEBS Letters (1996), 385 (1,2), 15-20CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
      A ribooligonucleotide duplex representing the acceptor stem of Escherichia coli tRNAAla with a G3-U70 wobble base pair, which is the main identity element for the recognition by the alanine-tRNA synthetase, has been characterized by 2D-NMR, as having two sequence variants with a regular Watson-Crick G3-C70 and an I3-U70 wobble pair, resp. As compared to a regular A-RNA, the G-U base pair gives rise to variations of the local helix geometry which are reflected in distinct local chem. shift changes. Structural differences between the duplex possessing an I3-U70 base pair and the wild-type G3-U70 sequence have also been found. The nucleotides in the ubiquitous single-stranded NCCA terminus display a surprisingly high degree of stacking order, esp. between A73, C74, and C75.
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      Hou, Y. M. and Schimmel, P. (1988) A simple structural feature is a major determinant of the identity of a transfer RNA Nature 333, 140– 145
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      A simple structural feature is a major determinant of the identity of a transfer RNA
      Hou, Ya Ming; Schimmel, Paul
      Nature (London, United Kingdom) (1988), 333 (6169), 140-5CODEN: NATUAS; ISSN:0028-0836.
      Anal. of a series of mutants of an Escherichia coli alanine tRNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine tRNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific tRNA.
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      McClain, W. H. and Foss, K. (1988) Changing the identity of a transfer RNA by introducing a G-U wobble pair near the 3′ acceptor end Science 240, 793– 796
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      Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end
      McClain, William H.; Foss, K.
      Science (Washington, DC, United States) (1988), 240 (4853), 793-6CODEN: SCIEAS; ISSN:0036-8075.
      Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of tRNA has remained unknown. To investigate the identity of a tRNA, the nucleotides at 3 computer-identified positions in tRNAPhe were replaced with the corresponding nucleotides from tRNAAla. The identity of the resulting tRNA, when examd. as an amber suppressor in Escherichia coli, was that of tRNAAla.
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    24. 24
      Mueller, U., Schubel, H., Sprinzl, M., and Heinemann, U. (1999) Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G·U wobble base pair at 1.16 angstrom resolution RNA 5, 670– 677
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      24
      Crystal structure of acceptor stem of tRNAAla from Escherichia coli shows unique G·U wobble base pair at 1.16 Å resolution
      Mueller, Uwe; Schubel, Harald; Sprinzl, Mathias; Heinemann, Udo
      RNA (1999), 5 (5), 670-677CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      The acceptor stem of Escherichia coli tRNAAla, rGGGGCUA·rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3-U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the crit. feature recognized by the synthetase. The authors here report the structure anal. of ALAwt at near-at. resoln. using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses. The two independent mols. in the asym. unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALAc70) detd. at 1.4 Å exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. The authors suggest that a highly conserved hydration pattern in both grooves around the G3·U70 wobble base pair may be functionally significant.
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      White, S. A. and Li, H. (1996) Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition RNA 2, 226– 234
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      Yeast ribosomal protein L32 recognizes an RNA G:U juxtaposition
      White, Susan A.; Li, Hu
      RNA (1996), 2 (3), 226-34CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      Yeast ribosomal protein L32, RPL32, specifically represses splicing by binding to a purine-rich asym. loop adjacent to the 5' splice site of its own transcript. A potential G:U pair closes the internal loop and the goal of the present study is to understand what features of the putative G:U pair are recognized by RPL32. Two RNA oligomers contg. 10 and 13 nt were annealed to form a bimol. stem-loop-stem protein-binding site. Protein binding to each of 16 sequence variants was examd. using electrophoretic bandshift and filter-binding expts. The protein binds to only the duplex RNA and not to the individual oligomers, and the G:U pair is crit. for full-strength binding. Mutation studies show that the duplex having a G:U has the highest protein affinity (Kd = 10 nM), followed by RNAs bearing G:A, C:C, U:A, U:C, or G:G. Duplexes contg. the other possible pairs bind very weakly and Watson-Crick pairing does not favor protein binding. The G of the G:U is required for strong protein binding, but replacement by inosine reduces binding only modestly. Therefore, the minor groove guanine amino group is not a key protein recognition element. Both nucleotides of the pair influence the binding strength, but their contributions are in general not additive. These data imply that the G:U is probably paired and influences binding indirectly through its effect on the conformation of the RNA.
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    26. 26
      Reyes, J. L., Gustafson, E. H., Luo, H. R., Moore, M. J., and Konarska, M. M. (1999) The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site RNA 5, 167– 179
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      26
      The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site
      Reyes, Jose L.; Gustafson, E. Hilary; Luo, Hongbo R.; Moore, Melissa J.; Konarska, Maria M.
      RNA (1999), 5 (2), 167-179CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      A U5 snRNP protein, hPrp8, forms a UV-induced crosslink with the 5' splice site (5'SS) RNA within splicing complex B assembled in trans- as well as in cis-splicing reactions. Both yeast and human Prp8 interact with the 5'SS, branch site, polypyrimidine tract, and 3'SS during splicing. To begin to define functional domains in Prp8 we have mapped the site of the 5'SS crosslink within the hPrp8 protein. Immunopptn. anal. limited the site of crosslink to the C-terminal 50-60-kDa segment of hPrp8. In addn., size comparison of the crosslink-contg. peptides generated with different proteolytic reagents with the pattern of fragments predicted from the hPrp8 sequence allowed for mapping of the crosslink to a stretch of five amino acids in the C-terminal portion of hPrp8 (positions 1894-1898). The site of the 5'SS:hPrp8 crosslink falls within a segment spanning the previously defined polypyrimidine tract recognition domain in yPrp8, suggesting that an overlapping region of Prp8 may be involved both in the 5'SS and polypyrimidine tract recognition events. In the context of other known interactions of Prp8, these results suggest that this protein may participate in formation of the catalytic center of the spliceosome.
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    27. 27
      Leung, S. S. and Koslowsky, D. J. (2001) Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of an mRNA bound to its cognate gRNA RNA 7, 1803– 1816
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      27
      Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of an mRNA bound to its cognate gRNA
      Leung, Sheldon S.; Koslowsky, Donna J.
      RNA (2001), 7 (12), 1803-1816CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      Posttranscriptional editing of trypanosome mitochondrial mRNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5' anchor, whereas its 3' U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examd. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examd. using soln.-probing techniques. The mRNA/gRNA crosslinked mols. are efficient substrates for gRNA-directed cleavage. In addn., when the cleavage assay is performed in the presence or absence of addnl. UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the mols. in a biol. relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide addnl. support for the predicted secondary structure of interacting gRNA/mRNA pairs.
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    28. 28
      Mooers, B. H. M. and Singh, A. (2011) The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate RNA 17, 1870– 1883
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      28
      The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate
      Mooers, Blaine H. M.; Singh, Amritanshu
      RNA (2011), 17 (10), 1870-1883CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 5' end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 3' ends of most guide RNAs have ∼15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G·U wobble base pairs. To gain insights into the structure of the U-helix, we crystd. 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 5'-AGA-3'/5'-UUU-3' base-pair triple. The fusion of two identical U-helixes head-to-head promoted crystn. We obtained X-ray diffraction data with a resoln. limit of 1.37 Å. The U-helix had low and high twist angles before and after each G·U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins.
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    29. 29
      Lu, K., Heng, X., Garyu, L., Monti, S., Garcia, E. L., Kharytonchyk, S., Dorjsuren, B., Kulandaivel, G., Jones, S., Hiremath, A., Divakaruni, S. S., LaCotti, C., Barton, S., Tummillo, D., Hosic, A., Edme, K., Albrecht, S., Telesnitsky, A., and Summers, M. F. (2011) NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging Science 334, 242– 245
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      29
      NMR detection of structures in the HIV-1 5'-leader RNA that regulate genome packaging
      Lu, Kun; Heng, Xiao; Garyu, Lianko; Monti, Sarah; Garcia, Eric L.; Kharytonchyk, Siarhei; Dorjsuren, Bilguujin; Kulandaivel, Gowry; Jones, Simonne; Hiremath, Atheeth; Divakaruni, Sai Sachin; LaCotti, Courtney; Barton, Shawn; Tummillo, Daniel; Hosic, Azra; Edme, Kedy; Albrecht, Sara; Telesnitsky, Alice; Summers, Michael F.
      Science (Washington, DC, United States) (2011), 334 (6053), 242-245CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a NMR approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are crit. for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.
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    30. 30
      Knitt, D. S., Narlikar, G. J., and Herschlag, D. (1994) Dissection of the role of the conserved G·U pair in group I RNA self-splicing Biochemistry 33, 13864– 13879
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      30
      Dissection of the Role of the Conserved G·U Pair in Group I RNA Self-Splicing
      Knitt, Deborah S.; Narlikar, Geeta J.; Herschlag, Daniel
      Biochemistry (1994), 33 (46), 13864-79CODEN: BICHAW; ISSN:0006-2960.
      Phylogenetic conservation among >100 group I introns and previous in vitro studies have implicated a G· U pair as defining the 5'-splice site for exon ligation. The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon. The authors now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA. The following results indicate that interactions with this G·U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chem. step. The oligonucleotide, CCCUCU, binds to the ribozyme ∼20-fold stronger than CCCUCC despite the fact that the U-contg. oligonucleotide forms an ∼5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG. This and two independent exptl. observations indicate that the G·U pair contributes ∼100-fold (3 kcal/mol, 50°) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The ∼50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G·U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites. Comparison of the rates of the chem. cleavage step with G·U vs. G·C suggests that the G·U pair contributes ∼10-fold to the chem. step. It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chem. transition state via an intramol. hydrogen bond. Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent x-ray crystallog. structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water mol., thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion. Finally, kinetic and thermodn. evidence for the formation of a G·C+ wobble pair is presented. The tertiary energy of recognition of the G·U wobble pair appears to be sufficient to perturb the pKa of C to favor a G·C+ wobble pair instead of the Watson-Crick pair, despite the loss of a hydrogen bond in the base pair. This provides an example of RNA tertiary structure detg. secondary structure.
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    31. 31
      Pyle, A. M., Moran, S., Strobel, S. A., Chapman, T., Turner, D. H., and Cech, T. R. (1994) Replacement of the conserved G·U with a G-C pair at the cleavage site of the tetrahymena ribozyme decreases binding, reactivity, and fidelity Biochemistry 33, 13856– 13863
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      31
      Replacement of the Conserved G·U with a G-C Pair at the Cleavage Site of the Tetrahymena Ribozyme Decreases Binding, Reactivity, and Fidelity
      Pyle, Anna Marie; Moran, Sean; Strobel, Scott A.; Chapman, Teresa; Turner, Douglas H.; Cech, Thomas R.
      Biochemistry (1994), 33 (46), 13856-63CODEN: BICHAW; ISSN:0006-2960.
      There is a phylogenetically conserved G·U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. The authors have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G·U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42°C was measured by native gel electrophoresis and equil. dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G·U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G·U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position. Taken together, the results suggest that a G-C pair at the ribozyme cleavage site makes docking of the guide-sequence-substrate helix into the catalytic site less favorable than a G·U pair. The resulting consequences of weaker binding, slower reaction, and reduced cleavage fidelity provide a rationale for the phylogenetic conservation of the G·U.
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    32. 32
      Strobel, S. A. and Cech, T. R. (1995) Minor groove recognition of the conserved G·U pair at the tetrahymena ribozyme reaction site Science 267, 675– 679
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      32
      Minor groove recognition of the conserved G·U pair at the Tetrahymena ribozyme reaction site
      Strobel, Scott A.; Cech, Thomas R.
      Science (Washington, D. C.) (1995), 267 (5198), 675-9CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The guanine-uracil (G·U) base pair that helps to define the 5'-splice site of group I introns is phylogenetically highly conserved. In such a wobble base pair, G makes 2 H-bonds with U in a geometry shifted from that of a canonical Watson-Crick pair. The contribution made by individual functional groups of the G.U pair in the context of the Tetrahymena ribozyme was examd. by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex. The substitutions demonstrate that the exocyclic amine of G, when presented on the minor groove surface by the wobble base pair conformation, contributes substantially (2 kcal.mole-1) to binding by making a tertiary interaction with the ribozyme active site. It contributes addnl. to transition state stabilization. The ribozyme active site also makes tertiary contacts with a tripod of 2'-hydroxyls on the minor groove surface of the splice site helix. This suggests that the ribozyme binds the duplex primarily in the minor groove. Alanyl-aminoacyl-tRNA synthetase recognizes the exocyclic amine of an invariant G.U pair and contacts a similar array of 2'-hydroxyls when binding the tRNAAla acceptor stem, providing an unanticipated parallel between protein-RNA and RNA-RNA interactions.
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    33. 33
      Strobel, S. A. and Cech, T. R. (1996) Exocyclic amine of the conserved G·U pair at the cleavage site of the Tetrahymena ribozyme contributes to 5′-splice site selection and transition state stabilization Biochemistry 35, 1201– 1211
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      33
      Exocyclic Amine of the Conserved G·U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and Transition State Stabilization
      Strobel, Scott A.; Cech, Thomas R.
      Biochemistry (1996), 35 (4), 1201-11CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      A phylogenetically conserved guanine·uracil (G·U) pair defines the 5'-exon/intron boundary of precursor RNAs contg. group I introns. In this wobble base pair, the G forms two hydrogen bonds with U in a base pairing geometry shifted from that of a canonical Watson-Crick pair. On the basis of thermodn. measurements of synthetic base pair analogs (inosine, diaminopurine riboside, guanosine, or adenosine paired with U, C, or isocytidine) in place of the G·U pair, the authors have previously reported that the N2 exocyclic amine of the G is important for docking the 5'-exon into the active site of the Tetrahymena ribozyme [Strobel, S. A., & Cech, T. R. (1995) Science 267, 675-679]. Here the authors describe kinetic characterization of ribozyme-substrate combinations contg. the same series of analogs. By measuring the rate consts. of 5'-exon miscleavage (cleavage at incorrect phosphates), the authors demonstrate that the 5'-exon/intron boundary is primarily defined by the exocyclic amine of the G. The amine makes its contribution (2.5 kcal·mol-1) in the context of all three wobble pairs tested but fails to make a significant contribution (<0.8 kcal·mol-1) when presented in a Watson-Crick base pairing geometry. The authors also demonstrate that the exocyclic amine makes a modest contribution to chem. transition state stabilization (1.0 kcal·mol-1 relative to an inosine-U pair). The majority of this transition state contribution (0.7 kcal·mol-1) is independent of that contributed by the 2'-hydroxyl of the neighboring U. This argues against the model in which substantial transition state stabilization is derived from a water mol. bridging between the exocyclic amine of G and the 2'-hydroxyl of U. Instead it suggests that the tertiary interaction between the exocyclic amine and its hydrogen bonding partner in the active site is slightly improved during the chem. transition. The authors conclude that the exocyclic amine of G is the primary contributor to many characteristics of reactivity that have been ascribed to the conserved G·U pair, including stabilization of the chem. transition state and definition of the 5'-exon/intron boundary.
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    34. 34
      Šponer, J., Šponer, J. E., Petrov, A. I., and Leontis, N. B. (2010) Quantum chemical studies of nucleic acids: Can we construct a bridge to the RNA structural biology and bioinformatics communities? J. Phys. Chem. B 114, 15723– 15741
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      34
      Quantum Chemical Studies of Nucleic Acids: Can We Construct a Bridge to the RNA Structural Biology and Bioinformatics Communities?
      Sponer, Jiri; Sponer, Judit E.; Petrov, Anton I.; Leontis, Neocles B.
      Journal of Physical Chemistry B (2010), 114 (48), 15723-15741CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      A review. In this feature article, the authors provide a side-by-side introduction for 2 research fields: quantum chem. calcns. of mol. interaction in nucleic acids and RNA structural bioinformatics. The authors' main aim is to demonstrate that these research areas, while largely sepd. in contemporary literature, have substantial potential to complement each other that could significantly contribute to the authors' understanding of the exciting world of nucleic acids. The authors identify research questions amenable to the combined application of modern ab initio methods and bioinformatics anal. of exptl. structures while also assessing the limitations of these approaches. The ultimate aim is to attain valuable physicochem. insights regarding the nature of the fundamental mol. interactions and how they shape RNA structures, dynamics, function, and evolution.
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    35. 35
      Chen, J.-H., Gong, B., Bevilacqua, P. C., Carey, P. R., and Golden, B. L. (2009) A catalytic metal ion interacts with the cleavage site G·U wobble in the HDV ribozyme Biochemistry 48, 1498– 1507
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      35
      A Catalytic Metal Ion Interacts with the Cleavage Site G·U Wobble in the HDV Ribozyme
      Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.
      Biochemistry (2009), 48 (7), 1498-1507CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The hepatitis delta virus (HDV) ribozyme self-cleaves by a chem. mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochem. studies from several labs. have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted >2 pH units toward neutrality, while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been obsd. in high-resoln. crystal structures of various reaction states and mutants. Herein, we use soln. kinetic expts. and parallel Raman crystallog. studies to examine the effects of pH on the rate and Mg2+ binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G·U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochem. data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochem. data.
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    36. 36
      Chen, J.-H., Yajima, R., Chadalavada, D. M., Chase, E., Bevilacqua, P. C., and Golden, B. L. (2010) A 1.9 Å crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage Biochemistry 49, 6508– 6518
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      36
      A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage
      Chen, Jui-Hui; Yajima, Rieko; Chadalavada, Durga M.; Chase, Elaine; Bevilacqua, Philip C.; Golden, Barbara L.
      Biochemistry (2010), 49 (31), 6508-6518CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pKa shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg2+ ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochem.; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystd. and detd. the structure of the HDV ribozyme bound to an inhibitor RNA contg. a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg2+ ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg2+ ion is also found within the active site where it interacts with a newly resolved G·U reverse wobble. Although the inhibitor exhibits crystallog. disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand obsd. in the hammerhead ribozyme. This model suggests that the pro-RP oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg2+ ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.
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    37. 37
      Keel, A. Y., Rambo, R. P., Batey, R. T., and Kieft, J. S. (2007) A general strategy to solve the phase problem in RNA crystallography Structure 15, 761– 772
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      A general strategy to solve the phase problem in RNA crystallography
      Keel, Amanda Y.; Rambo, Robert P.; Batey, Robert T.; Kieft, Jeffrey S.
      Structure (Cambridge, MA, United States) (2007), 15 (7), 761-772CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      X-ray crystallog. of biol. important RNA mols. has been hampered by tech. challenges, including finding heavy-atom derivs. to obtain high-quality exptl. phase information. Existing techniques have drawbacks, limiting the rate at which important new structures are solved. To address this, the authors have developed a reliable means to localize heavy atoms specifically to virtually any RNA. By solving the crystal structures of thirteen variants of the G·U wobble pair cation binding motif, the authors have identified a version that when inserted into an RNA helix introduces a high-occupancy cation binding site suitable for phasing. This "directed soaking" strategy can be integrated fully into existing RNA crystallog. methods, potentially increasing the rate at which important structures are solved and facilitating routine solving of structures using Cu-Kα radiation. This method already has been used to solve several crystal structures.
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    38. 38
      Kieft, J. S. and Tinoco, I. (1997) Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine Structure 5, 713– 721
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      Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine
      Kieft, Jeffrey S.; Tinoco, Ignacio, Jr.
      Structure (London) (1997), 5 (5), 713-721CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
      Solvated metal ions are crit. for the proper folding and function of RNA. Despite the importance of these ions, the details of specific metal ion-RNA interactions are poorly understood. The crystal structure of a group I intron ribozyme domain characterized several metal-binding sites in the RNA with osmium (III) hexammine bound in the major groove. A corresponding method for locating and characterizing metal-binding sites of RNA in soln. is of obvious interest. NMR should be ideal for localizing metal hexammine ions bound to the RNA because of the large concn. of protons around the metal center. We have solved the soln. structure of the P5b stem loop from a group I intron ribozyme bound to a cobalt (III) hexammine ion. The location of the ion is precisely detd. by intermol. nuclear Overhausser effect cross-peaks between the cobalt (III) hexammine protons and both exchangeable and non-exchangeable RNA protons in the major groove. The binding site consists of tandem G-U base pairs in a sequence of four consecutive G residues ending in a GAAA tetraloop, as originally identified in the crystal structure. The edges of the bases in the major groove present an electrostatically neg. face and a variety of hydrogen-bond acceptors for the cobalt (III) hexammine ion. The metal ion ligand is bound near the guanosine nucleotides of the adjacent G-U base pairs, where it makes hydrogen bonds with the N7 and carbonyl groups of both guanines. The carbonyl groups of the uracil residues add to the neg. surface of the binding pocket, but do not form hydrogen bonds with the hexammine. Addnl. hydrogen bonds form with other guanine residues of the GGGG sequence. The structure of the binding site does not change significantly on binding the cobalt (III) hexammine. The structure of the complex in soln. is very similar to the structure in the crystal. The structure presents a picture of how tandem G-U base pairs bind and position metal ions within the RNA major groove. The binding site is performed in the absence of metal ions, and presents a neg. pocket in the major groove with a variety of hydrogen-bond acceptors. Because G-U base pairs are such a common motif in RNA sequences, it is possible that this RNA-metal ion interaction is crit. in forming large complex RNA structures such as those found in the ribosome and self-splicing introns. This structure was detd. using cobalt (III) hexammine as an analog for hexahydrated magnesium, a technique that may be applicable to other RNA sequences. Metal hexammines may prove to be useful general probes for locating RNA metal ion binding sites in soln.
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      Wang, W. M., Zhao, J. W., Han, Q. W., Wang, G., Yang, G. C., Shallop, A. J., Liu, J., Gaffney, B. L., and Jones, R. A. (2009) Modulation of RNA metal binding by flanking bases: N-15 NMR evaluation of GC, tandem GU, and tandem GA sites Nucleosides Nucleotides Nucleic Acids 28, 424– 434
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      Modulation of RNA metal binding by flanking bases: 15N NMR evaluation of GC, tandem GU, and tandem GA sites
      Wang, Weimin; Zhao, Jianwei; Han, Qianwei; Wang, Gang; Yang, Gengcheng; Shallop, Anthony J.; Liu, Jane; Gaffney, Barbara L.; Jones, Roger A.
      Nucleosides, Nucleotides & Nucleic Acids (2009), 28 (5-7), 424-434CODEN: NNNAFY; ISSN:1525-7770. (Taylor & Francis, Inc.)
      15N NMR chem. shift changes in the presence of Mg(H2O)62+, Zn2+, Cd2+, and Co(NH3)63+ were used to probe the effect of flanking bases on metal binding sites in three different RNA motifs. We found that: for GC pairs, the presence of a flanking purine creates a site for the soft metals Zn2+ and Cd2+ only; a GG-UU motif selectively binds only Co(NH3)63+, while a UG-GU motif binds none of these metals; a 3' guanosine flanking the adenosine of a sheared GA·AG pair creates an unusually strong binding site that precludes binding to the cross-strand stacked guanosines within the tandem pair.
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    40. 40
      Colmenarejo, G. and Tinoco, I., Jr. (1999) Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme J. Mol. Biol. 290, 119– 135
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      40
      Structure and Thermodynamics of Metal Binding in the P5 Helix of a Group I Intron Ribozyme
      Colmenarejo, Gonzalo; Tinoco, Ignacio, Jr.
      Journal of Molecular Biology (1999), 290 (1), 119-135CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      The soln. structure of an RNA hairpin modeling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been detd. by NMR. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and pos. intermol. nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+mol. is in the major groove of tandem G·U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G·U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Neg. and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrns. of P5 show specific broadening of protons of the G·U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding consts. for the interaction of Co(NH3)63+and of Mg2+ to P5 were detd. by monitoring imino proton chem. shifts during titrn. of the RNA with the metal ions. Dissocn. consts. are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G·U base-pairs. The affinities of Co(NH3)63+and Mg2+ for the tandem G·U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G·C or A·U for G·U pairs also affected the binding, as expected. These structural and thermodn. results provide systematic new information about major groove metal ion binding in RNA. (c) 1999 Academic Press.
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      Gautheret, D., Konings, D., and Gutell, R. R. (1995) G·U base pairing motifs in ribosomal RNA RNA 1, 807– 814
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      G·U base pairing motifs in ribosomal RNA
      Gautheret, Daniel; Konings, Danielle; Gutell, Robin R.
      RNA (1995), 1 (8), 807-14CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      An increasing no. of recognition mechanisms in RNA are found to involve G.·U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G·U pairs. Approx. 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G·U pairs are consistent with previous proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G·U pairs in orientations that could cause severe stacking problems. In addn., three motifs displaying a conserved G·U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3'3'GU5', as well as G·U pairs flanked by a bulge loop 3' of U. The possible structures and functions of these recurrent motifs are discussed.
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      Gray, D. M. (1997) Derivation of nearest-neighbor properties from data on nucleic acid oligomers. 1. Simple sets of independent sequences and the influence of absent nearest neighbors Biopolymers 42, 783– 793
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      Derivation of nearest-neighbor properties from data on nucleic acid oligomers. I. Simple sets of independent sequences and the influence of absent nearest neighbors
      Gray, Donald M.
      Biopolymers (1997), 42 (7), 783-793CODEN: BIPMAA; ISSN:0006-3525. (John Wiley & Sons, Inc.)
      The constraints on combinations of nearest neighbors in nucleic acid sequences and the nos. of independent sequences needed to describe nearest-neighbor properties of oligomers and polymers are derived and summarized. It has been pointed out in previous work [D. M. Gray and I. Tinoco, Jr. (1970) Biopolymers, Vol. 9, pp. 223-244; R. F. Goldstein and A. S. Benight (1992) Biopolymers, Vol. 32, pp. 1679-1693] that these constraints restrict the information available from measurements of properties of sequence combinations. The emphasis in this paper is on the properties of oligomer sequences that vary in length, where each nucleotide or base pair at the end of the sequence makes a significant contribution to the measured property by interacting with its boundary of fixed sequence or solvent. In such cases it is not possible to det. values of properties of individual nearest neighbors, except for the like neighbors [e.g., d(A-A), d(G-G), d(T-T), and d(C-C) nucleotide neighbors in single-stranded DNA or d(A-A)/d(T-T) and d(G-G)/d(C-C) base pair neighbors in double-stranded DNA], solely from measurements of properties of different sequences. Even values for properties of the like neighbors cannot be detd. from such oligomeric sequences if the sequences are all of the same length. Nearest-neighbor properties of oligomer sequences that vary in length can be summarized in terms of the values for independent sets of sequences that are nearest neighbors and monomers all with boundaries of the fixed sequence or solvent. Straightforward combinations of the values for the independent sequences will give the values of the property for any dependent sequence, without explicit knowledge of the individual nearest-neighbor values. These considerations have important consequences for the derivation of widely used thermodn. parameters, as discussed in the following paper.
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      Nguyen, M.-T. and Schroeder, S. J. (2010) Consecutive terminal GU pairs stabilize RNA helices Biochemistry 49, 10574– 10581
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      Consecutive Terminal GU Pairs Stabilize RNA Helices
      Nguyen, Mai-Thao; Schroeder, Susan J.
      Biochemistry (2010), 49 (49), 10574-10581CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Consecutive GU pairs at the ends of RNA helixes provide significant thermodn. stability between -1.0 and -3.8 kcal/mol at 37°, which is equiv. to approx. 2 orders of magnitude in the value of a binding const. The thermodn. stabilities of GU pairs depend on the sequence, stacking orientation, and position in the helix. In contrast to GU pairs in the middle of a helix that may be destabilizing, all consecutive terminal GU pairs contribute favorable thermodn. stability. This work presents measured thermodn. stabilities for 30 duplexes contg. two, three, or four consecutive GU pairs at the ends of RNA helixes and a model to predict the thermodn. stabilities of terminal GU pairs. Imino proton NMR spectra show that the terminal GU nucleotides form hydrogen-bonded pairs. Different orientations of terminal GU pairs can have different conformations with equiv. thermodn. stabilities. These new data and prediction model will help improve RNA secondary structure prediction, identification of miRNA target sequences with GU pairs, and efforts to understand the fundamental phys. forces directing RNA structure and energetics.
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      Serra, M. J., Smolter, P. E., and Westhof, E. (2004) Pronouced instability of tandem IU base pairs in RNA Nucleic Acids Res. 32, 1824– 1828
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      Pronounced instability of tandem IU base pairs in RNA
      Serra, Martin J.; Smolter, Patricia E.; Westhof, Eric
      Nucleic Acids Research (2004), 32 (5), 1824-1828CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Optical melting was used to det. the stabilities of three series of RNA oligomers contg. tandem XU base pairs, GGCXUGCC (5'XU3'), GGCUXGCC (5'UX3') and GGCXXGGC/CCGUUCCG (5'XX3'), where X is either A, G or I (inosine). The helixes contg. tandem AU base pairs were the most stable in the first two series (5'XU3' and 5'UX3'), with an av. melting temp. ∼11° higher than the helixes with tandem 5'GU3' base pairs and 25° higher than the helixes with tandem 5'IU3' base pairs. For the third series (5'XX3'), the helix contg. tandem GG is the most stable, with an av. melting temp. ∼2° higher than the helix with tandem AA base pairs and ∼24° higher than the helix with tandem II base pairs. The thermodn. stability of the oligomers with tandem IU base pairs was also investigated as a function of magnesium ion concn. As with normal A-U or G-U tandem duplexes, the data could best be interpreted as non-specific binding of magnesium ions to the inosine-contg. RNA oligonucleotides.
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      Fukada, H. and Takahashi, K. (1998) Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride Proteins 33, 159– 166
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      Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride
      Fukada, Harumi; Takahashi, Katsutada
      Proteins: Structure, Function, and Genetics (1998), 33 (2), 159-166CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)
      Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically detd. in 0.1 M potassium chloride at temps. ranging from 5 to 45°. The values of the dissocn. const. were also detd. by means of potentiometric titrn. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temp. The enthalpy changes for the second dissocn. of phosphate and glycerol 2-phosphate where divalent anion is formed on dissocn. were fitted with the second order function of temp. rather than the first order. Temp. dependence of buffer pH calcd. by using the enthalpy and heat capacity changes obtained was in good agreement with the temp. variation of the pH values actually measured in the temp. range between 0 and 50° for all the buffers studied. On the basis of the results obtained, a numeric table showing the temp. dependence of pK values for the 18 buffers is presented.
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      Solvent suppression with symmetrically-shifted pulses
      Smallcombe, Stephen H.
      Journal of the American Chemical Society (1993), 115 (11), 4776-85CODEN: JACSAT; ISSN:0002-7863.
      Sym.-shifted shaped pulses are used to suppress large solvent resonances such as water while allowing the observation of protons exchanging with or barrier under the water resonance. Sym.-shifted pulses are a family of pulses that have been optimized for non-excitation of the water resonance and can be seen as having historical precedence in, and many of the properties of, long rectangular pulses and binomial sequences. Linear prediction software coupled with data shifting is used to eliminate the undesirable frequency-dependent phase and baseline problems normally assocd. with pulses or excitation schemes of this length. Zero-frequency solvent subtraction is also described. Results are illustrated for DNA and protein samples in 90% water.
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      Grzesiek, S. and Bax, A. (1993) The importance of not saturating H2O in protein NMR - application to sensitivity enhancement and NOE measurements J. Am. Chem. Soc. 115, 12593– 12594
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      The importance of not saturating water in protein NMR. Application to sensitivity enhancement and NOE measurements
      Grzesiek, Stephan; Bax, Ad
      Journal of the American Chemical Society (1993), 115 (26), 12593-4CODEN: JACSAT; ISSN:0002-7863.
      The longitudinal relaxation time, T1, of H2O is nearly four times longer than the av. T1 of protein protons. If the intense water signal is suppressed by means of purge pulses this results in a satd. state for the slowly relaxing H2O signal, which via cross relaxation attenuates the protein spectrum. It is demonstrated that by designing pulse sequences in such a way that the water remains close to its equil. value during the entire expt. a significant enhancement in sensitivity can be obtained, particularly for the resonances of rapidly exchanging amide protons. The new approach returns the water magnetization to the z axis at the start of every acquisition, thereby improving water suppression compared to techniques that scramble magnetization by presatn. or purging pulses. This approach is demonstrated for 1H-15N correlation of the backbone amides in a complex between calmodulin and a tightly binding 26-residue peptide fragment of skeletal muscle myosin light chain kinase. Avoiding satn. of the water resonance is also shown to be important in measuring NOE interactions between water and protein protons, and for quant. measurement of the heteronuclear 15N-(1H) NOE. This latter measurement indicates that calmodulin residues 75 through 80 in the calmodulin peptide complex are subject to substantial internal dynamics, whereas the amides of residues 75 through 82 are subject to fast hydrogen exchange.
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      Piotto, Martial; Saudek, Valdimir; Sklenar, Vladimir
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      A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aq. solns. (WATERGATE, WATER suppression by GrAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM soln. of a double-stranded DNA fragment in 90% H2O at 5° is presented.
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      Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: A multidimensional spectral processing system based on UNIX pipes J. Biomol. NMR 6, 277– 293
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      The NMRPipe system is a UNIX software environment of processing, graphics, and anal. tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
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      Investigation of the Structural Basis for Thermodynamic Stabilities of Tandem GU Mismatches: Solution Structure of (rGAGGUCUC)2 by Two-Dimensional NMR and Simulated Annealing
      McDowell, Jeffrey A.; Turner, Douglas H.
      Biochemistry (1996), 35 (45), 14077-14089CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The duplex (rGAGGUCUC)2 contains the motif 3'-CUGG-5'5'-GGUC-3', which is unusually stable compared with other sym. tandem GU mismatches and occurs in the P5 helix of the group I intron of Tetrahymena thermophila. The three-dimensional soln. structure of (rGAGGUCUC)2 was detd. using two-dimensional NMR and a simulated annealing protocol. The structure is remarkably similar to the A-DNA crystal structure of (dGGGGTCCC)2 [Kneale, G., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 186, 805-814] which contains the analogous motif 3'-CTGG-5'5'-GGTC-3'. Incorporation of the 3'-CUGG-5'5'-GGUC-3' motif has little effect on backbone torsion angles and helical parameters compared with std. A-form. The only significant departure from A-form is a slight overtwisting 5' of the G in the GU mismatch and a displacement of the mismatches toward the minor groove. Inspection of stacking patterns of this structure and comparison with sym. tandem GT mismatches in A-DNA oligonucleotides from crystal structure data suggest that electrostatics are important in detg. motif stability.
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      Progress in Nuclear Magnetic Resonance Spectroscopy (1996), 29 (1/2), 51-127CODEN: PNMRAT; ISSN:0079-6565. (Elsevier)
      A review with 268 refs. Topics discussed include: RNA synthesis and purifn.; NMR spectroscopy; spectral assignments; constraints for structure detn.; structure calcns.;.
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      Chaires, Jonathan B.
      Biophysical Chemistry (1997), 64 (1-3), 15-23CODEN: BICIAZ; ISSN:0301-4622. (Elsevier)
      Monte Carlo simulations were used to reveal a subtle, hidden contribution to van't Hoff enthalpy ests. from small heat capacity changes. In simulated van't Hoff plots of ln K vs. 1/T, small heat capacity changes (-200 ≤ ΔCp ≤ +200 cal mol-1 K-1), for which curvature could not be discerned within the noise of the data, were found to systematically bias the slope, leading to apparently erroneous enthalpy ests. Nonlinear least squares anal. of the simulated van't Hoff plots further revealed that it is difficult to ext. statistically reliable ΔCp values from data with even modest noise levels. Ests. of ΔCp and ΔHvH were found to be highly correlated, indicating an ill-posed nonlinear fitting problem. Nonlinear fits were found in many cases to be statistically no better than simpler linear fits. These simulations show, however, that if an independent calorimetric enthalpy est. is available, apparent discrepancies between that value and ests. derived from van't Hoff plots may be used to infer the existence of a small heat capacity change and its sign. By this procedure, apparent differences between van't Hoff and calorimetric enthalpy ests. may be interpreted and reconciled. An important conclusion from these studies is that such differences most likely result for statistical reasons, rather than from underlying phys. causes.
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      Oligonucleotides (2003), 13 (6), 515-537CODEN: OLIGAJ; ISSN:1545-4576. (Mary Ann Liebert, Inc.)
      A review. The Tm is defined as temp. of melting or, more accurately, as temp. of midtransition. This term is often used for nucleic acids (DNA and RNA, oligonucleotides, and polynucleotides). A thermal denaturation expt. dets. the stability of the secondary structure of a DNA or RNA and aids in the choice of the sequences for antisense oligomers or PCR primers. Beyond a simple numerical value (the Tm), a thermal denaturation expt., in which the folded fraction of a structure is plotted vs. temp., yields important thermodn. information. The authors present classic problems encountered during these expts. and attempt to demonstrate that a no. of useful pieces of information can be extd. from these exptl. curves.
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      Biopolymers (1998), 44 (3), 309-319CODEN: BIPMAA; ISSN:0006-3525. (John Wiley & Sons, Inc.)
      A review with 76 refs. The thermodn. of RNA secondary structure formation in small model systems provides a database for predicting RNA structure from sequence. Methods for making these measurements are reviewed with emphasis on optical methods and treatment of exptl. errors. Anal. of exptl. results in terms of simple nearest-neighbor models is presented. Some measured sequence dependences of non-Watson-Crick motifs are discussed.
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      Chembiochem : a European journal of chemical biology (2003), 4 (10), 936-62 ISSN:1439-4227.
      NMR spectroscopy is a powerful tool for studying proteins and nucleic acids in solution. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules, proteins, ions, and small ligands can be obtained for RNA molecules up to 100 nucleotides. This review provides insight into the resonance assignment methods that are the first and crucial step of all NMR studies, into the determination of base-pair geometry, into the examination of local and global RNA conformation, and into the detection of interaction sites of RNA. Examples of NMR investigations of RNA are given by using several different RNA molecules to illustrate the information content obtainable by NMR spectroscopy and the applicability of NMR techniques to a wide range of biologically interesting RNA molecules.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3svmtVChsA%253D%253D&md5=900e56059b2d912c5d66ae5d6f55c6fe
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      Reid, B. R., McCollumn, L., Ribeiro, N. S., Abbate, J., and Hurd, R. E. (1979) Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid Biochemistry 18, 3996– 4005
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      67
      Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid
      Reid, Brian R.; McCollumn, Lillian; Ribeiro, N. Susan; Abbate, Joseph; Hurd, Ralph E.
      Biochemistry (1979), 18 (18), 3996-4005CODEN: BICHAW; ISSN:0006-2960.
      The low-field H bond ring NH proton NMR spectra of several tRNAs related to yeast tRNAPhe were examd. Several resonances were sensitive to Mg2+ and temp., suggesting that they are derived from tertiary base pairs. These same resonances could not be attributed to cloverleaf base pairs, as shown by exptl. assignment and ring current shift calcn. of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals ≥6 tertiary base pairs involving ring NH hydrogen bonds, which are apparently responsible for the extra resonances obsd. in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs were obsd. at the same position in the spectrum.
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    68. 68
      Johnston, P. D. and Redfield, A. G. (1981) Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons Biochemistry 20, 1147– 1156
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      68
      Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons
      Johnston, Paul D.; Redfield, Alfred G.
      Biochemistry (1981), 20 (5), 1147-56CODEN: BICHAW; ISSN:0006-2960.
      Results directed primarily toward spectral assignment and nuclear spin dynamics are described for yeast tRNAPhe in 0.1M NaCl, pH 7. Mg2+ titrns. were performed. Changes in the spectrum occur for Mg2+/tRNA ratios of ∼2 and >10. Difference spectroscopy between 43 and 29° in zero Mg2+ concn., together with prior identification of the GU4 acceptor stem base pair, indicates early acceptor melting and is used to identify acceptor resonances. Transport of spin energy (spin diffusion) is described in tRNA together with a summary of relevant expts. A survey of nuclear Overhauser effects (NOE) between imino and arom. and amino protons is concluded, together with some recent conclusions based on Me NOE and expts. with tRNAs deuterated at the purine C8 position. Assignment of the imino NMR spectrum on the basis of these and previous data is reviewed and discussed in detail. Preliminary distance ests. based on the NOE for AU and GU4 base pairs are in reasonable agreement with the expected distances.
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      Cockerill, M. (1993) Not much to malign - Multalin 4.0 Trends Biochem. Sci. 18, 106– 107
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      Deigan, K. E., Li, T. W., Mathews, D. H., and Weeks, K. M. (2009) Accurate SHAPE-directed RNA structure determination Proc. Natl. Acad. Sci. U.S.A. 106, 97– 102
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      Accurate SHAPE-directed RNA structure determination
      Deigan, Katherine E.; Li, Tian W.; Mathews, David H.; Weeks, Kevin M.
      Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (1), 97-102CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Almost all RNAs can fold to form extensive base-paired secondary structures. Many of these structures then modulate numerous fundamental elements of gene expression. Deducing these structure-function relationships requires that it be possible to predict RNA secondary structures accurately. However, RNA secondary structure prediction for large RNAs, such that a single predicted structure for a single sequence reliably represents the correct structure, has remained an unsolved problem. Here, we demonstrate that quant., nucleotide-resoln. information from a SHAPE expt. can be interpreted as a pseudo-free energy change term and used to det. RNA secondary structure with high accuracy. Free energy minimization, by using SHAPE pseudofree energies, in conjunction with nearest neighbor parameters, predicts the secondary structure of deproteinized Escherichia coli 16S rRNA (>1300 nt) and a set of smaller RNAs (75-155 nt) with accuracies of up to 96-100%, which are comparable to the best accuracies achievable by comparative sequence anal.
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    71. 71
      Hart, J. M., Kennedy, S. D., Mathews, D. H., and Turner, D. H. (2008) NMR-assisted prediction of RNA secondary structure: Identification of a probable pseudoknot in the coding region of an R2 Retrotransposon J. Am. Chem. Soc. 130, 10233– 10239
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      71
      NMR-Assisted Prediction of RNA Secondary Structure: Identification of a Probable Pseudoknot in the Coding Region of an R2 Retrotransposon
      Hart, James M.; Kennedy, Scott D.; Mathews, David H.; Turner, Douglas H.
      Journal of the American Chemical Society (2008), 130 (31), 10233-10239CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      As the rate of functional RNA sequence discovery escalates, high-throughput techniques for reliable structural detn. are becoming crucial for revealing the essential features of these RNAs in a timely fashion. Computational predictions of RNA secondary structure quickly generate reasonable models but suffer from several approxns., including overly simplified models and incomplete knowledge of significant interactions. Similar problems limit the accuracy of predictions for other self-folding polymers, including DNA and peptide nucleic acid (PNA). The work presented here demonstrates that incorporating unassigned data from simple NMR expts. into a dynamic folding algorithm greatly reduces the potential folding space of a given RNA and therefore increases the confidence and accuracy of modeling. This procedure has been packaged into an NMR-assisted prediction of secondary structure (NAPSS) algorithm that can produce pseudoknotted as well as non-pseudoknotted secondary structures. The method reveals a probable pseudoknot in the part of the coding region of the R2 retrotransposon from Bombyx mori that orchestrates second-strand DNA cleavage during insertion into the genome.
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    72. 72
      Batey, R. T., Rambo, R. P., and Doudna, J. A. (1999) Tertiary motifs in RNA structure and folding Angew. Chem., Int. Ed. 38, 2327– 2343
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      Tertiary motifs in RNA structure and folding
      Batey, Robert T.; Rambo, Robert P.; Doudna, Jennifer A.
      Angewandte Chemie, International Edition (1999), 38 (16), 2327-2343CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      A review with 140 refs. RNA plays a crit. role in mediating every step of the cellular information transfer pathway from DNA-encoded genes to functional proteins. Its diversity of biol. functions stems from the ability of RNA to act as a carrier of genetic information and to adopt complex 3-dimensional folds that create sites for chem. catalysis. At.-resoln. structures of several large RNA mols., detd. by x-ray crystallog., have elucidated some of the means by which a global fold is achieved. Within these RNAs are tertiary structural motifs that enable the highly anionic double-stranded helixes to tightly pack together to create a globular architecture. Here, an overview of the structures of these motifs and their contribution to the organization of large, biol. active RNAs is presented. Base stacking, participation of the ribose 2'-OH groups in H-bonding interactions, binding of divalent metal cations, noncanonical base pairing, and backbone topol. all serve to stabilize the global structure of RNA and play crit. roles in guiding the folding process. Studies of the RNA-folding problem, which is conceptually analogous to the protein-folding problem, have demonstrated that folding primarily proceeds through a hierarchical pathway in which domains assemble sequentially. Formation of the proper tertiary interactions between these domains leads to discrete intermediates along this pathway. Advances in the understanding of RNA structure have facilitated improvements in the techniques that are utilized in modeling the global architecture of biol. interesting RNAs that have been resistant to at.-resoln. structural anal.
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    73. 73
      Varani, G. and McClain, W. H. (2000) The G·U wobble base pair: a fundamental building block of RNA structure crucial to RNA function in diverse biological systems EMBO Rep. 1, 18– 23
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      The G·U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems
      Varani, Gabriele; McClain, William H.
      EMBO Reports (2000), 1 (1), 18-23CODEN: ERMEAX; ISSN:1469-221X. (Oxford University Press)
      A review with 47 refs. The G·U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodn. stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G·U or A·U base pairs. The G·U wobble base pair also has unique chem., structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites contg. G·U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biol. processes.
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      Allawi, H. T. and SantaLucia, J. (1997) Thermodynamics and NMR of internal G·T mismatches in DNA Biochemistry 36, 10581– 10594
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      Thermodynamics and NMR of Internal G·T Mismatches in DNA
      Allawi, Hatim T.; SantaLucia, John, Jr.
      Biochemistry (1997), 36 (34), 10581-10594CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Thermodn. of 39 oligonucleotides with internal G·T mismatches dissolved in 1 M NaCl were detd. from UV absorbance vs. temp. profiles. These data were combined with literature values of six sequences to derive parameters for 10 linearly independent trimer and tetramer sequences with G·T mismatches and Watson-Crick base pairs. The G·T mismatch parameters predict ΔG°37, ΔH°, ΔS°, and TM with av. deviations of 5.1%, 7.5%, 8.0%, and 1.4°, resp. These predictions are within the limits of what can be expected for a nearest-neighbor model. The data show that the contribution of a single G·T mismatch to helix stability is context dependent and ranges from +1.05 kcal/mol for AGA/TTT to -1.05 kcal/mol for CGC/GTG. Several tests of the applicability of the nearest-neighbor model to G·T mismatches are described. Anal. of imino proton chem. shifts show that structural perturbations from the G·T mismatches are highly localized. One-dimensional NOE difference spectra demonstrate that G·T mismatches form stable hydrogen-bonded wobble pairs in diverse contexts. Refined nearest-neighbor parameters for Watson-Crick base pairs are also presented.
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    75. 75
      Cate, J. H. and Doudna, J. A. (1996) Metal-binding sites in the major groove of a large ribozyme domain Structure 4, 1221– 1229
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      75
      Metal-binding sites in the major groove of a large ribozyme domain
      Cate, Jamie H.; Doudna, Jennifer A.
      Structure (London) (1996), 4 (10), 1221-1229CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
      Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific 3-dimensional structures that confer activity. Here, the 2.8 Å crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 domain, provides the 1st detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold were examd. in detail. Heavy-atom derivs. used in the structure detn. revealed characteristics of some of the metal-binding sites in the P4-P6 domain. Although long-range RNA-RNA contacts within the mol. primarily involved the minor groove, Os hexaammine bound at 3 locations in the major groove. All 3 sites involved guanine and uracil nucleotides exclusively; 2 were formed by G·U wobble base pairs. In the native RNA, 2 of the sites were occupied by fully-hydrated Mg2+ ions. Sm3+ bound specifically to the RNA by displacing a Mg2+ in a region crit. to the folding of the entire domain. Bound at specific sites in the P4-P6 domain RNA, Os(III) hexaammine produced the high-quality heavy-atom deriv. used for structure detn. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivs. with hexaammine compds. The features of the obsd. metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G·U base pairs in rRNAs are Mg2+-binding sites.
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    76. 76
      Konforti, B. B., Abramovitz, D. L., Duarte, C. M., Karpeisky, A., Beigelman, L., and Pyle, A. M. (1998) Ribozyme catalysis from the major groove of group II intron domain 5 Mol. Cell 1, 433– 441
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      76
      Ribozyme catalysis from the major groove of group II intron domain 5
      Konforti, Boyana B.; Abramovitz, Dana L.; Duarte, Carlos M.; Karpeisky, Alex; Beigelman, Leonid; Pyle, Anna Marie
      Molecular Cell (1998), 1 (3), 433-441CODEN: MOCEFL ISSN:. (Cell Press)
      The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examd. We obsd. that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the crit. G (specifically N7 or O6) is essential for chem. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the crit. G could engage in crit. tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.
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    77. 77
      Adams, P. L., Stahley, M. R., Kosek, A. B., Wang, J., and Strobel, S. A. (2004) Crystal structure of a self-splicing group I intron with both exons Nature 430, 45– 50
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      77
      Crystal structure of a self-splicing group I intron with both exons
      Adams, Peter L.; Stahley, Mary R.; Kosek, Anne B.; Wang, Jimin; Strobel, Scott A.
      Nature (London, United Kingdom) (2004), 430 (6995), 45-50CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here the authors report the X-ray crystal structure (3.1-Å resoln.) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asym. positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.
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    78. 78
      Forconi, M., Sengupta, R. N., Piccirilli, J. A., and Herschlag, D. (2010) A rearrangement of the guanosine-binding site establishes an extended network of functional interactions in the tetrahymena group I ribozyme active site Biochemistry 49, 2753– 2762
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      78
      A Rearrangement of the Guanosine-Binding Site Establishes an Extended Network of Functional Interactions in the Tetrahymena Group I Ribozyme Active Site
      Forconi, Marcello; Sengupta, Raghuvir N.; Piccirilli, Joseph A.; Herschlag, Daniel
      Biochemistry (2010), 49 (12), 2753-2762CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Protein enzymes appear to use extensive packing and hydrogen bonding interactions to precisely position catalytic groups within active sites. Because of their inherent backbone flexibility and limited side chain repertoire, RNA enzymes face addnl. challenges relative to proteins in precisely positioning substrates and catalytic groups. Here, we use the group I ribozyme to probe the existence, establishment, and functional consequences of an extended network of interactions in an RNA active site. The group I ribozyme catalyzes a site-specific attack of guanosine on an oligonucleotide substrate. We previously detd. that the hydrogen bond between the exocyclic amino group of guanosine and the 2'-hydroxyl group at position A261 of the Tetrahymena group I ribozyme contributes to overall catalysis. We now use functional data, aided by double mutant cycles, to probe this hydrogen bond in the individual reaction steps of the catalytic cycle. Our results indicate that this hydrogen bond is not formed upon guanosine binding to the ribozyme but instead forms at a later stage of the catalytic cycle. Formation of this hydrogen bond is correlated with other structural rearrangements in the ribozyme's active site that are promoted by docking of the oligonucleotide substrate into the ribozyme's active site, and disruption of this interaction has deleterious consequences for the chem. transformation within the ternary complex. These results, combined with earlier results, provide insight into the nature of the multiple conformational steps used by the Tetrahymena group I ribozyme to achieve its active structure and reveal an intricate, extended network of interactions that is used to establish catalytic interactions within this RNA's active site.
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    79. 79
      Lipchock, S. V. and Strobel, S. A. (2008) A relaxed active site after exon ligation by the group I intron Proc. Natl. Acad. Sci. U.S.A 105, 5699– 5704
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      79
      A relaxed active site after exon ligation by the group I intron
      Lipchock, Sarah V.; Strobel, Scott A.
      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (15), 5699-5704CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two addnl. structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodn. measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.
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    80. 80
      Stahley, M. R., Adams, P. L., Wang, J., and Strobel, S. A. (2007) Structural metals in the group I intron: A ribozyme with a multiple metal ion core J. Mol. Biol. 372, 89– 102
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      80
      Structural Metals in the Group I Intron: A Ribozyme with a Multiple Metal Ion Core
      Stahley, Mary R.; Adams, Peter L.; Wang, Jimin; Strobel, Scott A.
      Journal of Molecular Biology (2007), 372 (1), 89-102CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We detd. the x-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addn. to 222 nucleotides of RNA, the model includes 18 Mg2+ and K+ ions. Five of the metals bind within 12 Å of the scissile phosphate and coordinate the majority of the oxygen atoms biochem. implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is crit. to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helixes. The group I intron has a rich history of biochem. efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochem. results to further understand the role of metal ions in group I intron structure and function.
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    81. 81
      Strobel, S. A. and Ortoleva-Donnelly, L. (1999) A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme Chem. Biol. 6, 153– 165
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      81
      A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme
      Strobel, Scott A.; Ortoleva-Donnelly, Lori
      Chemistry & Biology (1999), 6 (3), 153-165CODEN: CBOLE2; ISSN:1074-5521. (Current Biology Publications)
      The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chem. transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chem. basis of group I intron catalysis. Using nucleotide analog interference mapping and site-specific functional group substitution expts., we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chem. transition state by ∼ 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chem. step. These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.
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    82. 82
      Toor, N., Keating, K. S., Taylor, S. D., and Pyle, A. M. (2008) Crystal structure of a self-spliced group II intron Science 320, 77– 82
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      82
      Crystal Structure of a Self-Spliced Group II Intron
      Toor, Navtej; Keating, Kevin S.; Taylor, Sean D.; Pyle, Anna Marie
      Science (Washington, DC, United States) (2008), 320 (5872), 77-82CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Group II introns are self-splicing ribozymes that catalyze their own excision from precursor transcripts and insertion into new genetic locations. Here we report the crystal structure of an intact, self-spliced group II intron from Oceanobacillus iheyensis at 3.1 angstrom resoln. An extensive network of tertiary interactions facilitates the ordered packing of intron subdomains around a ribozyme core that includes catalytic domain V. The bulge of domain V adopts an unusual helical structure that is located adjacent to a major groove triple helix (catalytic triplex). The bulge and catalytic triplex jointly coordinate two divalent metal ions in a configuration that is consistent with a two-metal ion mechanism for catalysis. Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.
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    83. 83
      Xu, D., Landon, T., Greenbaum, N. L., and Fenley, M. O. (2007) The electrostatic characteristics of G·U wobble base pairs Nucleic Acids Res. 35, 3836– 3847
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      83
      The electrostatic characteristics of G•U wobble base pairs
      Xu, Darui; Landon, Theresa; Greenbaum, Nancy L.; Fenley, Marcia O.
      Nucleic Acids Research (2007), 35 (11), 3836-3847CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      G•U wobble base pairs are the most common and highly conserved non-Watson-Crick base pairs in RNA. Previous surface maps imply uniformly neg. electrostatic potential at the major groove of G•U wobble base pairs embedded in RNA helixes, suitable for entrapment of cationic ligands. In this work, we have used a Poisson-Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G•U wobble displays distinctly enhanced negativity compared with std. GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G•U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5kT/e, depending upon structural features. We propose that the negativity at the major groove of G•U wobble base pairs is detd. by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G•U sites in RNA are most likely to bind cationic ligands.
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    84. 84
      Chen, G., Znosko, B. M., Jiao, X. Q., and Turner, D. H. (2004) Factors affecting thermodynamic stabilities of RNA 3 × 3 internal loops Biochemistry 43, 12865– 12876
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      84
      Factors Affecting Thermodynamic Stabilities of RNA 3 × 3 Internal Loops
      Chen, Gang; Znosko, Brent M.; Jiao, Xiaoqi; Turner, Douglas H.
      Biochemistry (2004), 43 (40), 12865-12876CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Internal loops in RNA are important for folding and function. The 3 × 3 nucleotide internal loops are the smallest size sym. loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodn. and structural insights acquired for 3 × 3 loops should improve approxns. for stabilities of 3 × 3 and larger internal loops. Most natural 3 × 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides contg. different 3 × 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodn. stabilities of 3 × 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of ∼1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 × 3 loops is derived by multiple linear regression.
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    85. 85
      Serra, M. J., Baird, J. D., Dale, T., Fey, B. L., Retatagos, K., and Westhof, E. (2002) Effects of magnesium ions on the stabilization of RNA oligomers of defined structures RNA 8, 307– 323
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      Effects of magnesium ions on the stabilization of RNA oligomers of defined structures
      Serra, Martin J.; Baird, John D.; Dale, Taraka; Fey, Bridget L.; Retatagos, Kimberly; Westhof, Eric
      RNA (2002), 8 (3), 307-323CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      Optical melting was used to det. the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concn. The oligomers included helixes composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concn. on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temp. or free energy) was three times greater than that obsd. for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes obsd. upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes obsd. in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the obsd. cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calcd.
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    86. 86
      Walter, A. E., Wu, M., and Turner, D. H. (1994) The stability and structure of tandem GA mismatches in RNA depend on closing base-pairs Biochemistry 33, 11349– 11354
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      86
      The Stability and Structure of Tandem GA Mismatches in RNA Depend on Closing Base Pairs
      Walter, Amy E.; Wu, Ming; Turner, Douglas H.
      Biochemistry (1994), 33 (37), 11349-54CODEN: BICHAW; ISSN:0006-2960.
      UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal sequence loops in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chem. shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.
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    87. 87
      Freier, S. M., Burger, B. J., Alkema, D., Neilson, T., and Turner, D. H. (1983) Effects of 3′ dangling end stacking on the stability of GGCC and CCGG double helixes Biochemistry 22, 6198– 6206
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      87
      Effects of 3' dangling end stacking on the stability of GGCC and CCGG double helixes
      Freier, Susan M.; Burger, Barbara J.; Alkema, Dirk; Neilson, Thomas; Turner, Douglas H.
      Biochemistry (1983), 22 (26), 6198-206CODEN: BICHAW; ISSN:0006-2960.
      The thermodn. properties for helix formation of 2 core mols., GGCC and CCGG, and pentanucleotides contg. either core plus a 3'-dangling nucleotide were measured spectrophotometrically. In 1M Na+, the order of stability was: GGCC ≈ GGCCp < pGGCC < GGCCC ≈ GGCCCp < GGCCUp < GGCCAp ≈ GGCCGp ≈ GGCCεAp (where εA is 1,N6-ethenoadenosine); CCGG < pCCGG < CCGGCp < CCGGUp < CCGGAp ≈ CCGGGp. In 0.01M Na+, the order of stability for the GGCC family did not change except that GGCC was more stable than the tetramers with a terminal phosphate. Thermodn. parameters obtained by using a 2-state model demonstrated that the stabilizing effect of a 3'-dangling end was enthalpic. The results indicated that stacking is an important contributor to nucleic acid stability. Sedimentation equil. expts. at 3° on GGCCGp in 1M Na+ and on GGCCAp in 0.01M Na+ indicated no aggregation of pentanucleotide helixes at strand concns. as high as 2 mM.
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    88. 88
      Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., and Turner, D. H. (1985) Contributions of dangling end stacking and terminal base-pair formation to the stabilities of XGGCCp, XCCGGp, XGGCCYp, and XCCGGYp helixes Biochemistry 24, 4533– 4539
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      88
      Contributions of dangling end stacking and terminal base-pair formation to the stabilities of XGGCCp, XCCGGp, XGGCCYp, and XCCGGYp helixes
      Freier, Susan M.; Alkema, Dirk; Sinclair, Alison; Neilson, Thomas; Turner, Douglas H.
      Biochemistry (1985), 24 (17), 4533-9CODEN: BICHAW; ISSN:0006-2960.
      The role of stacking in terminal base-pair formation was studied by comparison of the stability increments for dangling ends to those for fully formed base pairs. Thermodn. parameters were measured spectrophotometrically for helix formation of the hexanucleotides, AGGCCUp, UGGCCAp, CGGCCGp, GCCGGCp, and UCCGGAp, and for the corresponding pentanucleotides contg. a 5'-dangling end on the GGCCp or CCGGp core helix. In 1M NaCl at 1 × 10-4 M strands, a 5'-dangling nucleotide in this series increased the duplex melting temp. (Tm) only 0-4°, about the same as adding a 5'-phosphate. In contrast, as detd. previously, a 3'-dangling nucleotide increased the Tm at 1 × 10-4 M strands 7-23°, depending on the sequence. These results were consistent with stacking patterns obsd. in A-form RNA. The stability increments from terminal A-U, C-G, or U-A base pairs on GGCC or a terminal U-A pair on CCGG were nearly equal to the sums of the stability increments from the corresponding dangling ends. This suggested that stacking plays a large role in nucleic acid stability. The stability increment from the terminal base pairs in GCCGGCp, however, was ∼5-fold the sum of the corresponding dangling ends, suggesting H-bonding can also make important contributions.
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    89. 89
      Crick, F. H. C. (1966) Codon-anticodon pairing: the wobble hypothesis J. Mol. Biol. 19, 548– 555
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      Codon-anticodon pairing: the wobble hypothesis
      Crick, F. H. C.
      Journal of Molecular Biology (1966), 19 (2), 548-55CODEN: JMOBAK; ISSN:0022-2836.
      It is suggested that while the standard base pairs may be used rather strictly in the 1st 2 positions of the triplet, there may be some wobble in the pairing of the third base. This hypothesis is explored systematically, and it is shown that such a wobble could explain the general nature of the degeneracy of the genetic code.
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    90. 90
      Chen, X. Y., McDowell, J. A., Kierzek, R., Krugh, T. R., and Turner, D. H. (2000) Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G·U pairs: One hydrogen bond for each G·U pair in r(GGCGUGCC)2 and two for each G·U pair in r(GAGUGCUC)2 Biochemistry 39, 8970– 8982
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      90
      Nuclear Magnetic Resonance Spectroscopy and Molecular Modeling Reveal That Different Hydrogen Bonding Patterns Are Possible for G·U Pairs: One Hydrogen Bond for Each G·U Pair in r(GGCGUGCC)2 and Two for Each G·U Pair in r(GAGUGCUC)2
      Chen, Xiaoying; McDowell, Jeffrey A.; Kierzek, Ryszard; Krugh, Thomas R.; Turner, Douglas H.
      Biochemistry (2000), 39 (30), 8970-8982CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      G·U pairs occur frequently and have many important biol. functions. The stability of sym. tandem G·U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G·U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G·U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochem. 34, 3204-3211]. In particular, at 37°, the motif 5'-CGUG-3' is less stable by approx. 3 kcal/mol compared with other sym. tandem G·U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The soln. structures of r(GAGUGCUC)2 and r(GGCGUGCC)2 duplexes have been detd. by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G·U pair region. The striking discovery is that in r(GGCGUGCC)2 each G·U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addn., the temp. dependence of the imino proton resonances is also consistent with the different no. of hydrogen bonds in the G·U pair. To test the NMR models, U or G in various G·U pairs were individually replaced by N3-methyluridine or isoguanosine, resp., thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.
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    91. 91
      Pan, Y. P., Priyakumar, U. D., and MacKerell, A. D. (2005) Conformational determinants of tandem GU mismatches in RNA: Insights from molecular dynamics simulations and quantum mechanical calculations Biochemistry 44, 1433– 1443
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      91
      Conformational Determinants of Tandem GU Mismatches in RNA: Insights from Molecular Dynamics Simulations and Quantum Mechanical Calculations
      Pan, Yongping; Priyakumar, U. Deva; MacKerell, Alexander D., Jr.
      Biochemistry (2005), 44 (5), 1433-1443CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Structure and energetic properties of base pair mismatches in duplex RNA have been the focus of numerous investigations due to their role in many important biol. functions. Such efforts have contributed to the development of models for secondary structure prediction of RNA, including the nearest-neighbor model. In RNA duplexes contg. GU mismatches, 5'-GU-3' tandem mismatches have a different thermodn. stability than 5'-UG-3' mismatches. In addn., 5'-GU-3' mismatches in some sequence contexts do not follow the nearest-neighbor model for stability. To characterize the underlying at. forces that det. the structural and thermodn. properties of GU tandem mismatches, mol. dynamics (MD) simulations were performed on a series of 5'-GU-3' and 5'-UG-3' duplexes in different sequence contexts. Overall, the MD-derived structural models agree well with exptl. data, including local deviations in base step helicoidal parameters in the region of the GU mismatches and the model where duplex stability is assocd. with the pattern of GU hydrogen bonding. Further anal. of the simulations, validated by data from quantum mech. calcns., suggests that the exptl. obsd. differences in thermodn. stability are dominated by GG interstrand followed by GU intrastrand base stacking interactions that dictate the one vs. two hydrogen bonding scenarios for the GU pairs. In addn., the inability of 5'-GU-3' mismatches in different sequence contexts to all fit into the nearest-neighbor model is indicated to be assocd. with interactions of the central four base pairs with the surrounding base pairs. The results emphasize the role of GG and GU stacking interactions on the structure and thermodn. of GU mismatches in RNA.
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    92. 92
      Biswas, R., Wahl, M. C., Ban, C., and Sundaralingam, M. (1997) Crystal structure of an alternating octamer r(GUAUGUA)dC with adjacent G·U wobble pairs J. Mol. Biol. 267, 1149– 1156
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      92
      Crystal structure of an alternating octamer r(GUAUGUA)dC with adjacent G·U wobble pairs
      Biswas, Roopa; Wahl, Markus C.; Ban, Changill; Sundaralingam, Muttaiya
      Journal of Molecular Biology (1997), 267 (5), 1149-1156CODEN: JMOBAK; ISSN:0022-2836. (Academic)
      The crystal structure of the RNA duplex, r(GUAUGUA)dC, with a 3'-terminal deoxy C residue, has been detd. at 1.38 Å resoln. The r(GUAUGU) hexameric consensus sequence is present at the exon-intron junction in pre-mRNAs of yeast and higher eukaryotic organisms. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 39.71 Å, c = 68.15 Å and γ = 120°C with one duplex in the asym. unit. The structure was solved using the mol. replacement method. The final model contains 332 atoms of the duplex and 67 solvent mols. The R-factor is 17.6% (Rfree of 23.1%) for 4035 reflections with F ≥ 1.5 σ(F) in the resoln. range 10.0 to 1.38 Å. The duplex is of the A-type with a pseudodyad relating the two strands. The RNA helix is slightly distorted, in spite of the presence of two adjacent G·U wobble base-pairs located at the center of the helix. The twist angle between the wobble pairs, 38.1°, is above the av. value and those between the wobble base-pairs and the flanking Watson-Crick base-pairs, 26.7° and 26.3°, resp., are lower than the av. values. The twist between the junction base-pairs are about 24°. The G·U wobble pairs are bridged by water mols. and solvated in the grooves. G·U base-pairs are as stable as the Watson-Crick A·U pairs and only slightly less stable than the G·C pairs accounting for their frequent occurrence in RNA.
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    93. 93
      Utsunomiya, R., Suto, K., Balasundaresan, D., Fukamizu, A., Kumar, P. K. R., and Mizuno, H. (2006) Structure of an RNA duplex r(GGCG(Br)UGCGCU)2 with terminal and internal tandem G·U base pairs Acta Crystallogr. D 62, 331– 338
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      93
      Structure of an RNA duplex r(GGCGBrUGCGCU)2 with terminal and internal tandem G.U base pairs
      Utsunomiya, Ryuji; Suto, Kyoko; Balasundaresan, Dhakshnamoorthy; Fukamizu, Akiyoshi; Kumar, Penmetcha K. R.; Mizuno, Hiroshi
      Acta Crystallographica, Section D: Biological Crystallography (2006), D62 (3), 331-338CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      The crystal structure of a self-complementary RNA duplex r(GGCGBrUGCGCU)2 with terminal G.U and internal tandem G.U base pairs has been detd. at 2.1 Å resoln. The crystals belong to the tetragonal space group P43, with unit-cell parameters a = b = 37.69, c = 96.28 Å and two duplexes in the asym. unit. The two strands of each duplex are related by a pseudodyad axis. The structure was refined to final Rwork and Rfree values of 20.9 and 25.3%, resp. The duplexes stack in an end-to-end manner, forming infinite columns along the c axis. This is the first structural study of an RNA duplex contg. G.U pairs at the termini. The stacking overlaps of the terminal G.U base pairs with their adjacent Watson-Crick base pairs are larger than those of Watson-Crick base pairs of the 5'-YR-3'/3'-RY-5' type. The terminal G.U base pairs of neighboring duplexes are also stacked with each other. An alternating underwound-overwound pattern of the twist angles is seen at each step along the duplex. This observation is typical for internal tandem G.U pairs, while the terminal G.U base pairs exhibit high twist angles with the adjacent Watson-Crick pairs. The 3'-side of U of the internal G.U base pair, which is unstacked, appears to be stabilized by π-cation interaction with an Mg2+ ion.
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    94. 94
      Biswas, R. and Sundaralingam, M. (1997) Crystal structure of r(GUGUGUA)dC with tandem G·U/U·G wobble pairs with strand slippage J. Mol. Biol. 270, 511– 519
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      94
      Crystal structure of r(GUGUGUA)dC with tandem G·U/U·G wobble pairs with strand slippage
      Biswas, Roopa; Sundaralingam, Muttaiya
      Journal of Molecular Biology (1997), 270 (3), 511-519CODEN: JMOBAK; ISSN:0022-2836. (Academic)
      To better understand the frequent occurrence of adjacent wobble pairs in rRNAs we have detd. the crystal structure of the RNA duplex, r(GUGUGUA)dC with the 3'-terminal deoxy C residue. Two different crystal forms of the duplex were obtained and both belong to the rhombohedral space group, R3. Crystal form I has hexagonal unit cell dimensions, a = b = 40.82 Å and c = 66.09 Å and diffracts to 1.58 Å resoln., while crystal form II has a = b = 47.11 Å and c = 59.86 Å, diffracting only to 2.50 Å resoln. Both structures were solved by the mol. replacement method using different starting models. In spite of the large differences in the cell dimensions the overall structures in both crystals are similar. Instead of the expected blunt-end duplex with four consecutive G·U pairs, the slippage of the strands resulted in two different tandem G·U/U·G wobble pairs involving two of the central and two of the 5' overhang bases, still yielding a total of four wobble pairs. These tandem wobble pairs are flanked by two Watson-Crick pairs. The A-type duplexes stack in the familiar head-to-tail fashion forming a pseudocontinuous helix. The wobble pairs of the present motif II (G·U/U·G) structure stack with a low twist angle of 25.3° in contrast to that of motif I (U·G/G·U), 38.1°. The four wobble pairs are characteristically heavily hydrated in both the grooves accounting for their stability.
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    95. 95
      McDowell, J. A., He, L. Y., Chen, X. Y., and Turner, D. H. (1997) Investigation of the structural basis for thermodynamic stabilities of tandem GU wobble pairs: NMR structures of (rGGAGUUCC)2 and (rGGAUGUCC)2 Biochemistry 36, 8030– 8038
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      95
      Investigation of the Structural Basis for Thermodynamic Stabilities of Tandem GU Wobble Pairs: NMR Structures of (rGGAGUUCC)2 and (rGGAUGUCC)2
      McDowell, Jeffrey A.; He, Liyan; Chen, Xiaoying; Turner, Douglas H.
      Biochemistry (1997), 36 (26), 8030-8038CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The sym., tandem GU mismatch motifs, 3'-UGUA-5'5'-AUGU-3' and 3'-UUGA-5'5'-AGUU-3', which only differ in the mismatch order, have an av. difference in thermodn. stability of 2 kcal/mol at 37°. Thermodn. studies of duplexes contg. these motifs indicate the effect is largely localized to the mismatches and adjacent base pairs. The three-dimensional structures of two representative duplexes, (rGGAGUUCC)2 and (rGGAUGUCC)2, were detd. by two-dimensional NMR and a simulated annealing protocol. Local deviations are similar to other intrahelical GU mismatches with little effect on backbone torsion angles and a slight overtwisting between the base pair 5' of the G of the mismatch and the mismatch itself. Comparisons of the resulting stacking patterns along with electrostatic potential maps suggest that interactions between highly neg. electrostatic regions between base pairs may play a role in the obsd. thermodn. differences.
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    96. 96
      Masquida, B. and Westhof, E. (2000) On the wobble G·U and related pairs RNA 6, 9– 15
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      96
      On the wobble GoU and related pairs
      Masquida, Benoit; Westhof, Eric
      RNA (2000), 6 (1), 9-15CODEN: RNARFU; ISSN:1355-8382. (Cambridge University Press)
      A review, with ∼60 refs. The wobble GoU pairs have been implicated in several biol. processes where RNA mols. play a key role. We review the geometrical and conformational properties of wobble GoU pairs on the basis of available crystal structures of RNAs at high resoln. The similarities with the wobble A+oC pairs and UoU pairs are illustrated, while the differences with the recently discovered bifurcated GU pairs are contrasted.
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    97. 97
      Jang, S. B., Hung, L. W., Jeong, M. S., Holbrook, E. L., Chen, X. Y., Turner, D. H., and Holbrook, S. R. (2006) The crystal structure at 1.5 Å resolution of an RNA octamer duplex containing tandem G·U basepairs Biophys. J. 90, 4530– 4537
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      97
      The crystal structure at 1.5 Å resolution of an RNA octamer duplex containing tandem G.U basepairs
      Jang, Se Bok; Hung, Li-Wei; Jeong, Mi Suk; Holbrook, Elizabeth L.; Chen, Xiaoying; Turner, Douglas H.; Holbrook, Stephen R.
      Biophysical Journal (2006), 90 (12), 4530-4537CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The crystal structure of the RNA octamer, 5'-GGCGUGCC-3' has been detd. from x-ray diffraction data to 1.5 Å resoln. In the crystal, this oligonucleotide forms five self-complementary double-helixes in the asym. unit. Tandem 5'GU/3'UG basepairs comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation among the five crystallog. duplexes and the soln. structure. The G.U pairs in the five duplexes of the crystal form two direct hydrogen bonds and are stabilized by water mols. that bridge between the base of guanine (N2) and the sugar (O2') of uracil. This contrasts with the NMR structure in which only one direct hydrogen bond is obsd. for the G.U pairs. The reduced stability of the r(CGUG)2 motif relative to the r(GGUC)2 motif may be explained by the lack of stacking of the uracil bases between the Watson-Crick and G.U pairs as obsd. in the crystal structure.
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    98. 98
      Deng, J. P. and Sundaralingam, M. (2000) Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G·G/U·U tandem wobble base pairs: comparison with other tandem G·U pairs Nucleic Acids Res. 28, 4376– 4381
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      98
      Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G.G/U.U tandem wobble base pairs: comparison with other tandem G.U pairs
      Deng, Junpeng; Sundaralingam, Muttaiya
      Nucleic Acids Research (2000), 28 (21), 4376-4381CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      We have detd. the crystal structure of the RNA octamer duplex r(guguuuac)/r(guaggcac) with a tandem wobble pair, G·G/U·U (motif III), to compare it with U·G/G·U (motif I) and G·U/U·G (motif II) and to better understand their relative stabilities. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 41.92 Å, c = 56.41 Å, and γ = 120°, with one duplex in the asym. unit. The structure was solved by the mol. replacement method at 1.9 Å resoln. and refined to a final R factor of 19.9% and Rfree of 23.3% for 2862 reflections in the resoln. range 10.0-1.9 Å with F ≥ 2σ(F). The final model contains 335 atoms for the RNA duplex and 30 water mols. The A-RNA stacks in the familiar head-to-tail fashion forming a pseudo-continuous helix. The uridine bases of the tandem U·G pairs have slipped towards the minor groove relative to the guanine bases and the uridine O2 atoms form bifurcated hydrogen bonds with the N1 and N2 of guanines. The N2 of guanine and O2 of uridine do not bridge the 'locked' water mol. in the minor groove, as in motifs I and II, but are bridged by water mols. in the major groove. A comparison of base stacking stabilities of motif III with motifs I and II confirms the result of thermodn. studies, motif I > motif III > motif II.
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    99. 99
      Shi, K., Wahl, M. C., and Sundaralingam, M. (1999) Crystal structure of an RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U base pairs Nucleic Acids Res. 27, 2196– 2201
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      99
      Crystal structure of an RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U base pairs
      Shi, Ke; Wahl, Markus; Sundaralingam, Muttaiya
      Nucleic Acids Research (1999), 27 (10), 2196-2201CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2 with non-adjacent G·U and U·G wobble pairs sepd. by four Watson-Crick base pairs has been detd. to 2.5 A resoln. Crystals belong to the space group R3; a = 33.09 Å, α = 87.30 with a pseudodyad related duplex in the asym. unit. The structure was refined to a final Rwork of 17.5% and Rfree of 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3'-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3' penultimate phosphates are obsd. As expected, the wobble pairs are displaced with guanine toward the minor groove and uracil toward the major groove. The largest twist angles (37.70 and 40.57°) are at steps G1·C17/G2·U16 and U7·G11/C8·G10, while the smallest twist angles (28.24 and 27.27°) are at G2·U16/G3·C15 and C6·G12/U7·G11 and conform to the pseudo-dyad symmetry of the duplex. The mol. has two unequal kinks (17 and 11°) at the wobble sites and a third kink at the central G5 site which may be attributed to trans α(O5'-P), trans γ(C4'-C5') backbone conformations. The 2'-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water mols.
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    100. 100
      Alvarez-Salgado, F., Desvaux, H., and Boulard, Y. (2006) NMR assessment of the global shape of a non-labelled DNA dodecamer containing a tandem of G·T mismatches Magn. Reson. Chem. 44, 1081– 1089
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      100
      NMR assessment of the global shape of a non-labelled DNA dodecamer containing a tandem of G-T mismatches
      Alvarez-Salgado, Francisco; Desvaux, Herve; Boulard, Yves
      Magnetic Resonance in Chemistry (2006), 44 (12), 1081-1089CODEN: MRCHEG; ISSN:0749-1581. (John Wiley & Sons Ltd.)
      We have carried out a soln. study of two non-labeled self-complementary DNA dodecamers d(GACTGTACAGTC)2 and d(GACTGTGCAGTC)2 by NMR, the second sequence composed of two G-T mismatches. Structures were detd. using distances extd. from NOE effects alone or using both NOE and RDC constraints, measured in three different liq. cryst. media. We ensured that our data on the influence of the mesogen on the DNA structures, and the way in which the RDCs were incorporated as constraints in the protocol refinement, were consistent. We also tested the influence of different sets of RDCs and the best means of optimizing the calcn. of Da and R. Resoln. and accuracy of the ten best energy final structures were compared. The addn. of a small set of RDC constraints significantly improves the final detd. structures. We took advantage of the specificity of the RDC, i.e. it contains orientational information, and explored the global shape of the DNA duplexes; it was found that the duplexes do not have a large curvature. For the G-T base pair, we obsd., in this particular sequence (tandem of G-T mismatches), a new pattern of base pairing, which involved the formation of a bifurcated hydrogen bond.
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    101. 101
      Sugimoto, N., Kierzek, R., Freier, S. M., and Turner, D. H. (1986) Energetics of internal GU mismatches in ribooligonucleotide helixes Biochemistry 25, 5755– 5759
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      101
      Energetics of internal GU mismatches in ribooligonucleotide helixes
      Sugimoto, Naoki; Kierzek, Ryszard; Freier, Susan M.; Turner, Douglas H.
      Biochemistry (1986), 25 (19), 5755-9CODEN: BICHAW; ISSN:0006-2960.
      Thermodn. parameters of helix formation were measured spectroscopically for 16 oligoribonucleotides contg. either internal GU mismatches or the corresponding AU pairs. Internal GU mismatches stabilize each helix, but not as much as the corresponding AU paris. The differences in the enthalpy and entropy changes of helix formation assocd. with replacing AU pairs with GU mismatches are less than previously realized. At both 25 and 37°, the decrease in helix stability assocd. with replacing an AU with a GU is also less than thought previously. Approxns. are suggested for predicting the effects of GU mismatches on helix stability.
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    102. 102
      Freier, S. M., Kierzek, R., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Free energy contributions of G·U and other terminal mismatches to helix stability Biochemistry 25, 3209– 3213
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      102
      Free energy contributions of G·U and other terminal mismatches to helix stability
      Freier, Susan M.; Kierzek, Ryszard; Caruthers, Marvin H.; Neilson, Thomas; Turner, Douglas H.
      Biochemistry (1986), 25 (11), 3209-13CODEN: BICHAW; ISSN:0006-2960.
      Thermodn. parameters of helix formation were measured spectroscopically for 7 hexaribonucleotides contg. a GC tetramer core and G·U or other terminal base-pair mismatches. The free energies of helix formation were compared with those for the tetramer core alone and with those for the hexamer with 6 Watson-Crick base pairs. In 1M NaCl at 37°, the free energy of a terminal G·U mismatch is about equal to that of the corresponding A·U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to the free energy (37°) for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G·U mismatches and G·C pairs suggest that when stacking is weak, the addnl. H bond in the G·C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.
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    103. 103
      Testa, S. M., Disney, M. D., Turner, D. H., and Kierzek, R. (1999) Thermodynamics of RNA-RNA duplexes with 2-or 4-thiouridines: Implications for antisense design and targeting a group I intron Biochemistry 38, 16655– 16662
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      103
      Thermodynamics of RNA-RNA Duplexes with 2- or 4-Thiouridines: Implications for Antisense Design and Targeting a Group I Intron
      Testa, Stephen M.; Disney, Matthew D.; Turner, Douglas H.; Kierzek, Ryszard
      Biochemistry (1999), 38 (50), 16655-16662CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Antisense compds. are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compd. and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s2U or s4U) show that s2U can increase the stability of base pairing with A more than with G, while s4U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s4U-G pair. To test the effects of s2U and s4U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs2U), and r(AUGACs4U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compds. targeting cellular RNA.
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    104. 104
      He, L., Kierzek, R., SantaLucia, J., Walter, A. E., and Turner, D. H. (1991) Nearest-neighbor parameters for G·U mismatches - 5′GU3′/3′UG5′ is destabilizing in the contexts CGUG/GUGC, UGUA/AUGU, and AGUU/UUGU but stabilizing in GGUC/CUGG Biochemistry 30, 11124– 11132
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      104
      Nearest-neighbor parameters for G·U mismatches: 5'GU3'/3'UG5' is destabilizing in the contexts CGUG/GUGC, UGUA/AUGU, and AGUU/UUGA but stabilizing in GGUC/CUGG
      He, Liyan; Kierzek, Ryszard; SantaLucia, John, Jr.; Walter, Amy E.; Turner, Douglas H.
      Biochemistry (1991), 30 (46), 11124-32CODEN: BICHAW; ISSN:0006-2960.
      Thermodn. parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides contg. G·U mismatches. The results are used to det. nearest-neighbor parameters for helix propagation by G·U mismatches. Surprisingly, the 5'GU3'/3'UG5' nearest-neighbor free energy increment is unfavorable in the contexts CGUG/GUGC'/UGUA/AUGU', and AGUU/UUGA but favorable in the context GGUC/CUGG'. This is a non-nearest-neighbor effect. In contrast, the 5'UG3'/3'GU5' free energy increment is favorable and independent of context. CD and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G·U mismatches have two slowly exchanging imino protons. The imino resonances for the G·U mismatches in GGAGU UCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temp. than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G·U mismatches in GGAUGUCC remain sharp at high temp. The improved parameters for G·U mismatches should improve predictions of RNA structure from sequence.
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    105. 105
      Xia, T. B., McDowell, J. A., and Turner, D. H. (1997) Thermodynamics of nonsymmetric tandem mismatches adjacent to G·C base pairs in RNA Biochemistry 36, 12486– 12497
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      105
      Thermodynamics of nonsymmetric tandem mismatches adjacent to G·C base pairs in RNA
      Xia, Tianbing; McDowell, Jeffrey A.; Turner, Douglas H.
      Biochemistry (1997), 36 (41), 12486-12497CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The thermodn. stabilities and structures of a series of RNA duplexes contg. nonsym. tandem mismatches in the context of 5'GAGXYGAG3'/3'CUCWZCUC5', where XY/WZ are tandem mismatches, were studied by UV melting and imino proton NMR. The contribution of one mismatch to the free energy increment for tandem mismatch formation depends on the identity of the other mismatch. Imino proton NMR indicates that this is partly because the structure of a mismatch is dependent on the adjacent mismatch. The results suggest that differences in size, shape, and hydrogen bonding of the adjacent mismatches play important roles in detg. loop stability. A model for predicting stabilities of all possible tandem mismatches is proposed based on these and previous results.
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    106. 106
      Sugimoto, N., Kierzek, R., and Turner, D. H. (1987) Sequence dependence for the energetics of terminal mismatches in ribonucleic acid Biochemistry 26, 4559– 4562
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      106
      Sequence dependence for the energetics of terminal mismatches in ribooligonucleotides
      Sugimoto, Naoki; Kierzek, Ryszard; Turner, Douglas H.
      Biochemistry (1987), 26 (14), 4559-62CODEN: BICHAW; ISSN:0006-2960.
      Stability increments of terminal mismatches on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 15 oligoribonucleotides contg. the core sequences and various mismatches. Free energy increments for mismatches in this series range from -0.5 to -1.1 kcal/mol. These increments for mismatches on AU base pairs are smaller than those measured previously on GC base pairs. The terminal GU mismatches in the sequences GAUGCAUUp and UAUGCAUGp add approx. the same stability increment as the corresponding terminal AU mismatch. The stability increments for pyrimidine-pyrimidine and pyrimidine-purine mismatches can be approximated within -0.3 kcal/mol by adding the stability increments for the corresponding 3' and 5' unpaired nucleotides (dangling ends). Stability increments for purine-mismatches are approximated well by the stability increment for the corresponding 3' dangling end made more favorable by 0.2 kcal/mol. These approxns. are used to provide a table of stability increments for all 48 possible sequences of mismatches.
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    107. 107
      Ziomek, K., Kierzek, E., Biala, E., and Kierzek, R. (2002) The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides Biophys. Chem. 97, 233– 241
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      107
      The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides
      Ziomek, Krzysztof; Kierzek, Elzbieta; Biala, Ewa; Kierzek, Ryszard
      Biophysical Chemistry (2002), 97 (2-3), 233-241CODEN: BICIAZ; ISSN:0301-4622. (Elsevier Science B.V.)
      The presence of various modifications within oligomers changes their thermodn. stability. To get more systematic data, we measured effects of 5- and 6-substituted uridine on thermal stability of (AUCUMod.AGAU)2 and (AUCUAGAUMod.)2. Collected results lead to the following conclusions: (i) 5-halogenated and 5-alkylated substituents of the uridine affect thermal stability of the RNA duplexes differently. Moreover, the 5-fluorouridine changes stability of the RNA duplexes opposite to remaining 5-halogenouridines; (ii) for oligomers contg. 5-chloro, 5-bromo or 5-iodouridine stronger hydrogen bond formed between oxygen-4 of the 5-halogenated uracil and 6-amino group of the adenine is presumably responsible for stabilizing effect; (iii) placing of A-U5R base pairs closer to the end of the duplex enhance thermal stability relatively to oligomer with central position of this base pair; (iv) the effects of 5-substituents are additive, particularly for substituents which stabilize RNA duplexes; (v) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine present at internal position of A-UMod. inhibit duplexes formation; (vi) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine placed as terminal base pairs stabilize the duplexes mostly via 3'-dangling end effect.
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    108. 108
      Schroeder, S. J. and Turner, D. H. (2001) Thermodynamic stabilities of internal loops with GU closing pairs in RNA Biochemistry 40, 11509– 11517
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      108
      Thermodynamic Stabilities of Internal Loops with GU Closing Pairs in RNA
      Schroeder, Susan J.; Turner, Douglas H.
      Biochemistry (2001), 40 (38), 11509-11517CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodn. parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes contg. small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodn. stabilities of asym. internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for sym. internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.
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    109. 109
      Schroeder, S. J. and Turner, D. H. (2000) Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA Biochemistry 39, 9257– 9274
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      109
      Factors Affecting the Thermodynamic Stability of Small Asymmetric Internal Loops in RNA
      Schroeder, Susan J.; Turner, Douglas H.
      Biochemistry (2000), 39 (31), 9257-9274CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Optical melting expts. were used to det. the thermodn. parameters for oligoribonucleotides contg. small asym. internal loops. The results show a broad range of thermodn. stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR expts. provide evidence for possible hydrogen bonding in GA and UU mismatches in some asym. loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodn. stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 × 3 and 1 × 3 internal loops.
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    110. 110
      Freier, S. M., Sinclair, A., Neilson, T., and Turner, D. H. (1985) Improved free energies for G·C base-pairs J. Mol. Biol. 185, 645– 647
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      110
      Improved free energies for G·C base-pairs
      Freier, Susan M.; Sinclair, Alison; Neilson, Thomas; Turner, Douglas H.
      Journal of Molecular Biology (1985), 185 (3), 645-7CODEN: JMOBAK; ISSN:0022-2836.
      Thermodn. parameters of helix formation are reported for 7 oligoribonucleotides contg. only G·C pairs. These data are used with the nearest-neighbor model to calc. enthalpies and free energies of base pair formation for G·C pairs. For helix initiation, the free energy change at 37°, ΔG037, is +3.9 kcal/mol; for helix propagation, the ΔG037 values are -2.3, -3.2, and -3.3 kcal/mol for C-G, G-G, and G-C neighbors, resp.
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    111. 111
      Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Improved free-energy parameters for predictions of RNA duplex stability Proc. Natl. Acad. Sci. U.S.A. 83, 9373– 9377
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      111
      Improved free-energy parameters for predictions of RNA duplex stability
      Freier, Susan M.; Kierzek, Ryszard; Jaeger, John A.; Sugimoto, Naoki; Caruthers, Marvin H.; Neilson, Thomas; Turner, Douglas H.
      Proceedings of the National Academy of Sciences of the United States of America (1986), 83 (24), 9373-7CODEN: PNASA6; ISSN:0027-8424.
      Thermodn. parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize exptl. errors in ΔG° at 37°. These parameters predict melting temps. of most oligonucleotide duplexes within 5°. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G·U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.
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    112. 112
      Freier, S. M., Sugimoto, N., Sinclair, A., Alkema, D., Neilson, T., Kierzek, R., Caruthers, M. H., and Turner, D. H. (1986) Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids Biochemistry 25, 3214– 3219
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      112
      Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids
      Freier, Susan M.; Sugimoto, Naoki; Sinclair, Alison; Alkema, Dirk; Neilson, Thomas; Kierzek, Ryszard; Caruthers, Marvin H.; Turner, Douglas H.
      Biochemistry (1986), 25 (11), 3214-19CODEN: BICHAW; ISSN:0006-2960.
      The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers, XGCGCp and GCGCYp, contg. the GCGC core plus a dangling end (X and Y are various nucleosides). Each 5' dangling end increases helix stability at 37° ∼0.2 kcal/mol and each 3' end increases helix stability 0.8-1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported previously for the GGCC core, indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G·C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of stacking and pairing. If pairing contributions are due to H bonding, the results suggest stacking and H bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment assocd. with forming a H bond is estd. to be -1 kcal/mol H bond.
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    113. 113
      Sugimoto, N., Kierzek, R., and Turner, D. H. (1987) Sequence dependence for the energetics of dangling ends and terminal base pairs in ribooligonucleotides Biochemistry 26, 4554– 4558
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      113
      Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid
      Sugimoto, Naoki; Kierzek, Ryszard; Turner, Douglas H.
      Biochemistry (1987), 26 (14), 4554-8CODEN: BICHAW; ISSN:0006-2960.
      Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides contg. the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the addnl. stability from a 3' dangling end on an AU base pair is less than that on a GC base pair. The sequence dependence provides a test for the importance of dangling ends for various RNa interactions. Correlations are suggested with codon context effects and with the 3-dimensional structure of yeast phenylalanine tRNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is assocd. with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA mols. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment assocd. with forming a H bond can be about -1 kcal/mol of H bond.
      >> More from SciFinder ®
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    114. 114
      Burkard, M. E. and Turner, D. H. (2000) NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs Biochemistry 39, 11748– 11762
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      114
      NMR Structures of r(GCAGGCGUGC)2 and Determinants of Stability for Single Guanosine-Guanosine Base Pairs
      Burkard, Mark E.; Turner, Douglas H.
      Biochemistry (2000), 39 (38), 11748-11762CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')2, in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, resp. An addnl. hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-Rp phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')2, the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodn. data for a variety of duplexes contg. pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')2, suggesting similar GG conformations.
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    115. 115
      Petersheim, M. and Turner, D. H. (1983) Base-stacking and base-pairing contributions to helix stability: thermodynamics of double-helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp Biochemistry 22, 256– 263
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      115
      Base-stacking and base-pairing contributions to helix stability: thermodynamics of double-helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp
      Petersheim, Matthew; Turner, Douglas H.
      Biochemistry (1983), 22 (2), 256-63CODEN: BICHAW; ISSN:0006-2960.
      The thermodn. of double-helix formation in 1M NaCl were measured spectrophotometrically for CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp. The results indicate that addnl. double-helical stability is conferred by the terminal unpaired bases. The 3'-adenine stabilizes the double-helix more than the 5'-adenine or the 3'-uracil. The increased stability is due to a more favorable enthalpy change for double-helix formation. Comparison of the thermodn. for CCGG, ACCGGp, CCGGUp, and ACCGGUp indicates that stacking interactions are somewhat more important than pairing interactions in detg. the stability of the terminal AU base pairs in ACCGGUp.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXkvVylug%253D%253D&md5=69930c02708fde075e62983178ec135a
    116. 116
      Kierzek, R., Caruthers, M. H., Longfellow, C. E., Swinton, D., Turner, D. H., and Freier, S. M. (1986) Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability Biochemistry 25, 7840– 7846
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      116
      Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability
      Kierzek, Ryszard; Caruthers, Marvin H.; Longfellow, Carl E.; Swinton, David; Turner, Douglas H.; Freier, Susan M.
      Biochemistry (1986), 25 (24), 7840-6CODEN: BICHAW; ISSN:0006-2960.
      Solid-phase method using a phosphoramidite approach was used for synthesis of oligoribonucleotide pairs of oligomers with identical nearest neighbors but different sequences. Comparison of thermodn. parameters for these pairs provides a test of the nearest-neighbor hypothesis for predication of helix stability. In general, pairs of sequences with identical nearest neighbors have enthalpy and entropy changes for helix formation that differ by 8% on av., ΔG°37 that differ by 6% on av., and melding temps. within 0-5° of each other. These limits are typical of the accuracy that should be expected from nearest-neighbor predictions of RNA helix stability. UCAUGA and UGAUCA have the nearest neighbors but melting temps. that differ by 7°. This suggests some sequences will not be approximated well by the nearest-neighbor model.
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    117. 117
      Kierzek, R., Burkard, M. E., and Turner, D. H. (1999) Thermodynamics of single mismatches in RNA duplexes Biochemistry 38, 14214– 14223
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      117
      Thermodynamics of Single Mismatches in RNA Duplexes
      Kierzek, Ryszard; Burkard, Mark E.; Turner, Douglas H.
      Biochemistry (1999), 38 (43), 14214-14223CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The thermodn. properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR expts. The free energy increments at 37 °C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approxns. are suggested for predicting stabilities of single mismatches in short RNA duplexes.
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  • Supporting Information

    Supporting Information

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    (I) Thermodynamic parameters for duplex formation of Watson–Crick sequences. (II) Experimental thermodynamic parameters and error limits for newly measured sequences. (III) Component free energies and enthalpies of GU pairs. (IV) Free energies of doublets and triplets containing GU pairs calculated as component ΔG°37 of their sequences. (V) Free energy differences between sequences where GC pair(s) were replaced by AU pair(s). (VI) INN parameters for GU pairs calculated with a separate term for terminal GU pairs. (VII) Probability density function of the Student’s t-distribution for each INN motif with a separate parameter for terminal GU pairs. (VIII) 2D NOESY spectra for r(AGGCUU)2 showing H2′, H1′, and H6/H8 regions. (IX) 2D NOESY spectra for r(AUGCGU)2 showing H2′, H1′, and H6/H8 regions. (X) Desalting procedure for oligoribonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org.


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