Synthesis of a cleavable dinucleotide photoaffinity probe of ribonucleic acid polymerase: application to trinucleotide labeling of an Escherichia coli transcription complex

Abstract

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.