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Article

Structural Features and Thermodynamics of the J4/5 Loop from the Candida albicans and Candida dubliniensis Group I Introns^†^,^‡

Supporting Info

Brent M. Znosko,^§ Scott D. Kennedy, Pamela C. Wille,^§ Thomas R. Krugh,^§ and Douglas H. Turner*^§^#

Department of Chemistry, RC Box 270216, Department of Biochemistry and Biophysics, School of Medicine and Dentistry, and Department of Pediatrics and Center for Human Genetics and Molecular Pediatric Disease, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642

Biochemistry, 2004, 43 (50), pp 15822–15837

DOI: 10.1021/bi049256y

Publication Date (Web): November 23, 2004

†

This work was supported by NIH Grants GM 22939 (to D.H.T.) and GM 53826 (to T.R.K.).

‡

Protein Data Bank entry 1TUT.

Department of Chemistry.

Current address: Department of Chemistry, St. Louis University, St. Louis, MO 63103-2010.

Department of Biochemistry and Biophysics.

To whom correspondence should be addressed. Phone: (585) 275-3207. Fax: (585) 276-0205. E-mail: Turner@chem.rochester.edu.

Department of Pediatrics and Center for Human Genetics and Molecular Pediatric Disease.

Abstract

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5‘GAAGG3‘/3‘UAAUU5‘, potentially contains two A·A pairs flanked by one G·U pair on one side and two G·U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A·A pairs with a cross-strand stack and only the G·U pair not adjacent to an A·A pair forming a static wobble G·U pair. The two G·U pairs adjacent to the tandem A·A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH₃)₆³⁺ stabilizes the loop by several kilocalories per mole at 37 °C, addition of Mg²⁺ or Co(NH₃)₆³⁺ has no effect on the structure of the loop. The tandem G·U pairs provide a pocket of negative charge for Co(NH₃)₆³⁺ to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G·U pairs adjacent to internal loops.