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

Abstract

A cleavable dinucleotide reagent was prepared and used to map the path of the leading end of the RNA transcript across the surface of E. coli RNA polymerase/phage 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 RNA 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 labeled the DNA and the .beta. and .beta.'' subunits of RNA polymerase, with > 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 2 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 RNA longer than the trinucleotide.