Photoaffinity labeling of Escherichia coli RNA polymerase/poly[d(A-T)] transcription complexes by nascent RNA

Abstract

To elucidate the molecular interactions during transcription by Escherichia coli RNA polymerase, we have performed a quantitative analysis of the photoaffinity labeling produced by an aryl azide positioned at the leading (5') end of the nascent RNA. Macromolecular contacts on the path of RNA across the transcription complex containing the template poly[d(A-T)] are observed as a function of the length of the transcript. Quantitative analysis provides the percent yield of photoaffinity labeling in the transcription complex by each length of RNA. Significant yields are observed for DNA, the beta/beta' subunits (analyzed together), and the sigma subunit. The alpha subunit is not labeled under these experimental conditions. The DNA template is labeled by the leading ends of RNA molecules 5-18 bases long, with yields ranging from 1% to 6%. Photoaffinity labeling of poly[d(A-T)] is also observed for many transcript lengths longer than 18 nucleotides, but the yields are too low to quantitate. Labeling of the beta/beta' subunits occurs with approximately 50% yields for transcripts of lengths greater than or equal to 12 nucleotides; low but significant labeling yields of 1-8% by shorter RNAs (3-10 nucleotides) are observed. Labeling of the sigma subunit is detectable for transcripts from 7 to more than 19 nucleotides long; quantitative measurements were possible up to the 19-mer. The RNAs most likely to be photoattached to the sigma subunit are 9-12 nucleotides long, with a maximum photoaffinity labeling yield of 15% by the decanucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic acid‐binding regions of the second‐largest subunit of Drosophila RNA polymerase II identified by Southwestern blotting

“…and o were labeled [9]. However, no DNA binding can be observed for the B subunit alone, consistent with our results from Southwestern assays, but sub-assembled a&3 complexes bind to limited sites on DNA [ 13,14,24]. The observation that most of the known mutations responsible for rifampicin resistance and streptolydigin resistance resides in a region of the E. colip subunit corresponding to nucleic acid-binding region 1 points to a functional importance of this region.…”

Effects of an anti-.alpha. monoclonal antibody on interaction of Escherichia coli RNA polymerase with lac promoters

“…Apart from its involvement in the formation of the RNA polymerase core the role of the a subunit has not been defined. Data obtained from cross-linking studies have not provided evidence for contacts between a and DNA (Chenchick et al, 1981;Park et al, 1982) or between a and the nascent transcript (Bernhard & Meares, 1986;Stackhouse & Meares, 1988). The anti-a monoclonal antibody, mAb 126C6, used in this study has been shown to bind to an epitope that is available on both of the a subunits in the RNA polymerase core and holoenzyme (Riftina et al, 1989).…”

Chemical and photochemical probing of DNA complexes

“…In parallel approaches using chemical or photochemical probes attached to oligonucleotide primers for RNA polymerase, Grachev et al (1989) and Stackhouse andMeares (1988, 1989) have very elegantly probed the contacts between the growing nascent RNA chain and the E. coli RNA polymerase. Similar experiments with yeast RNA polymerase have also been performed (Riva et ul., 1987).…”