Topography of transcription: path of the leading end of nascent RNA through the Escherichia coli transcription complex.

Abstract

A cleavable dinucleotide photoaffinity reagent was prepared and used to map the path of the leading end of the RNA transcript across the surface of Escherichia coli RNA polymerase/T7 DNA transcription complexes. By using 5'-(4-azidophenacylthio)phosphoryladenylyl(3'-5')uridine, transcription was specifically initiated at the A1 promoter of bacteriophage T7 D111 or D123 DNA. Transcription complexes containing radiolabeled RNA chains of various lengths (4-116 nucleotides) were prepared, and the 5' end of the RNA transcript was then covalently attached to the nearby polymerase subunits or DNA by irradiation with UV light. The photoaffinity-labeled enzyme subunits and DNA were separated, the radiolabeled RNAs were cleaved from each, and the lengths and sequences of RNA attached to each component were determined. The leading end of RNA chains up to 12 bases long was found to label the DNA and the beta and beta' subunits of RNA polymerase, with more than 90% of the label going to the DNA. When the RNA transcript reached 12 bases in length, the 5' end diverged from the DNA and only the beta and beta' enzyme subunits were labeled thereafter. These two subunits were heavily labeled by RNA chains 12 to as many as 94 bases long. No significant labeling of the alpha subunit occurred. The sigma subunit was not labeled by RNAs longer than the trinucleotide.