Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analog. 1. Initiation and pyrophosphate exchange reactions

Abstract

The diastereomers of adenosine 5''-O-(1-thiotriphosphate) (ATP.alpha.S) and adenosine 5''-O-(2-thiotriphosphate) (ATP.beta.S) can replace ATP in the initiation reaction catalyzed by DNA dependent RNA polymerase from E. coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diastereomers of 3''-uridyl 5''-adenosyl O,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase, but the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP.alpha.S, isomer A, as substrate, the stereochemical courses of both the initiation and primed initiation reactions, respectively, were determined with [phage] T7 DNA template and they proceeded with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.