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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Topography of transcription: path of the leading end of nascent RNA through the Escherichia coli transcription complex. 619 29

The cleavable dinucleotide photoaffinity probe 5'-[[(4-azidophenacyl)thio]phosphoryl]adenylyl(3'-5')uridine was prepared and used to determine the 5' contacts of a trinucleotide in an Escherichia coli RNA polymerase/T7 DNA transcription complex. The probe was prepared by alkylating 5'-(thiophosphoryl)adenylyl(3'-5')uridine with azidophenacyl bromide. The 5'-(thiophosphorylyl(3'-5')uridine was prepared by the abortive initiation reaction of RNA polymerase on a poly[d(A-T)] DNA template, using adenosine 5'-O-(thiomonophosphate) and uridine triphosphate as substrates. A transcription complex containing a radiolabeled trinucleotide at the A1 promoter of bacteriophage T7 D111 or D123 DNA was prepared by using the dinucleotide photoaffinity probe as initiator and cytidine [alpha-32P]triphosphate as the other substrate. After photolysis, the labeled subunits and DNA were isolated, and the trinucleotide was removed in the presence of phenylmercuric acetate and analyzed by polyacrylamide gel electrophoresis. The 5' end of the trinucleotide was found to label the DNA (approximately 88%) and also the beta (approximately 10%) and sigma (approximately 3%) subunits of E. coli RNA polymerase. It was also shown that the order of migration of the beta and beta' subunits of E. coli RNA polymerase on polyacrylamide gel electrophoresis in sodium dodecyl sulfate is different from that in sodium dodecyl sulfate plus urea.
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PMID:Synthesis of a cleavable dinucleotide photoaffinity probe of ribonucleic acid polymerase: application to trinucleotide labeling of an Escherichia coli transcription complex. 635 6