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 β/β′ subunits (analyzed together), and the σ subunit. The α 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 β/β′ subunits occurs with ≈50% yields for transcripts of lengths ≥12 nucleotides; low but significant labeling yields of 1-8% by shorter RNAs (3-10 nucleotides) are observed. Labeling of the σ 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 σ subunit are 9-12 nucleotides long, with a maximum photoaffinity labeling yield of 15% by the decanucleotide. These results modify the conclusions of previous work concerning the release of σ from an E. coli RNA polymerase/poly[d(A-T)] transcription complex [Hansen, U. M., & McClure, W. R. (1980) J. Biol. Chem. 255, 9564-9570]. The photoaffinity labeling of σ in poly[d(A-T)] transcription complexes differs from the results observed with DNA containing either the λ P_R or the T7 A1 promoter [Bernhard, S. L., & Meares, C. F. (1986) Biochemistry 25, 5914-5919], providing further evidence that the interaction between the nucleic acids and the σ subunit in the transcription complex depends on the nucleotide sequence.