Photoaffinity labeling of Escherichia coli RNA polymerase/poly[d(A-T)] transcription complexes by nascent RNA.
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)
Stackhouse
T M
TM
Chemistry Department, University of California, Davis 95616.
Meares
C F
CF
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D011067:Poly dA-dT / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D011089:Polydeoxyribonucleotides; D012329:RNA, Bacterial; D011067:Poly dA-dT; D012321:DNA-Directed RNA Polymerases