Bernhard S L, Meares C F
Biochemistry. 1986 Oct 21;25(21):6397-404. doi: 10.1021/bi00369a009.
A photoaffinity-protection technique has been developed to study the accessibility of the leading (5') end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA. The macromolecules contacted by the leading (5') end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA [Hanna, M. M., & Meares, C. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4238-4242]. By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length. We examined in detail RNA molecules containing 11-50 nucleotides, whose 5' ends label the beta and beta' enzyme subunits with good yield. The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to beta beta' in thiol-treated samples to controls not treated with thiol. Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36% of the 5'-azides on RNAs 11-13 bases long and approximately 43% on RNAs 28-37 bases long but practically none of the 5'-azides on RNAs 40-43 bases long. Also notable was the reduction of 34 +/- 1% of the 5'-azides on RNA 12 bases long but only 14 +/- 2% on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.
