A heteroduplex template circumvents the energetic requirement for ATP during activated transcription by RNA polymerase II

作者：Dean R Tantin, Michael Fionn Carey · 发表于：Journal of Biological Chemistry · 年份：1994 · DOI：10.1016/s0021-9258(17)32452-3 · 被引用次数：60 · 研究领域：DNA and Nucleic Acid Chemistry、RNA Interference and Gene Delivery、RNA and protein synthesis mechanisms

Unlike most eukaryotic and prokaryotic RNA polymerases, promoter-specific transcription by RNA polymerase I1 requires hydrolysis of the ATP p-y phosphoanhydride bond.Here we show that a template containing a 10-base pair DNA mismatch encompassing the start site circumvents this requirement such that the non-hydrolyzable ATP analogues, ATPyS (adenosine 5'-O-(thiotriphosphate)) and AMF"PNP (adenyl-5"yl imidodiphosphate), support both basal and GAU-VPlG-activated transcription in a reconstituted HeLa cell in vitro transcription system.The results imply that ATP regulates either opening of the template at the start site or a closely associated step.Initiation of transcription in prokaryotic and eukaryotic cells proceeds through a multistep process: formation of a stable complex between RNA polymerase and double-stranded promoter DNA(the closed complex), melting of the start site to expose the template strand (the open complex), initiation of transcription, and promoter clearance (1).Most RNA polymerases apparently do not require exogenous energy to execute these steps.Eukaryotic RNA polymerase I1 (pol 11)' is somewhat unique in this regard because efficient transcription in vitro requires a hydrolyzable ATP P-y phosphoanhydride bond (2).The nonhydrolyzable ATP analogue AMP-PNP cannot substitute for ATP during pol I1 transcription from either the adenovirus major late (AdML) or early IV region (E41 promoters in a HeLa cell extract.Other y-imido derivatized nucleotides (Le.GMP-PNP)...