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Probing the Protein−DNA Contacts of a Yeast RNA Polymerase III Transcription Complex in a Crude Extract:  Solid Phase Synthesis of DNA Photoaffinity Probes Containing a Novel Photoreactive Deoxycytidine Analog

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Department of Medical Biochemistry, School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-4413
Cite this: Biochemistry 1996, 35, 30, 9821–9831
Publication Date (Web):July 30, 1996
https://doi.org/10.1021/bi960525x
Copyright © 1996 American Chemical Society

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    Abstract

    A novel photoreactive deoxycytidine analog, 4-[N-(p-azidobenzoyl)-2-aminoethyl]-dCTP (AB-dCTP), has been synthesized and incorporated at specific sites within the SUP4 tRNATyr gene. Immobilized single-stranded DNA was annealed to specific oligonucleotides and AB-dCMP incorporated into DNA by primer extension. DNA photoaffinity labeling with AB-dCMP was used to survey protein−DNA contacts in initiation and elongation complexes of RNA polymerase III (Pol III), and compared to DNA photoaffinity labeling using the previously described photoreactive deoxyuridine analog, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP (AB-dUMP) [Bartholomew et al. (1993) Mol. Cell.Biol. 13, 942−952]. In contrast to previous studies, we have used a crude protein fraction rather than highly purified preparations of Pol III and transcription factors TFIIIC and TFIIIB to examine if some component of the transcription complex is lost upon purification. Eleven nucleotide positions from bp −17 to bp +17 (+1 being the start site of transcription) on the nontranscribed strand were modified and shown to have little or no effect on transcription complex formation, initiation, or elongation as determined by multiple-round transcription assays. Efficient photoaffinity labeling by DNA containing AB-dCMP gave results comparable to that with AB-dUMP at proximal nucleotide positions and provided new evidence for the placement of the 160 and 31 kDa subunits of Pol III near the 5‘ end of the transcriptional bubble in an elongation complex. A novel 40 kDa protein was cross-linked at bps −17, −9, and −8 in a TFIIIC-dependent manner that had not been previously detected.

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    This work was supported by Public Health Service Grant GM 48413-02 from the National Institute of General Medical Sciences, National Institutes of Health.

     Children's Memorial Hospital, 2300 Childrens Plaza, Box 203 CMIER, Chicago, IL 60614.

    *

     To whom correspondence should be addressed. Phone:  (618)-453-6437/6436. Fax:  (618)-453-6440. E-mail:  [email protected].

     Abstract published in Advance ACS Abstracts, July 1, 1996.

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