Dvir A, Garrett K P, Chalut C, Egly J M, Conaway J W, Conaway R C
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, 73104, USA.
J Biol Chem. 1996 Mar 29;271(13):7245-8. doi: 10.1074/jbc.271.13.7245.
A requirement for an ATP cofactor in synthesis of the first 8-10 bonds of promoter-specific transcripts by RNA polymerase II is well established. Whether ATP is required for synthesis of the first phosphodiester bond or at a slightly later stage in synthesis of nascent transcripts, however, remains controversial. Goodrich and Tjian (Goodrich, J.A., and Tjian, R. (1994) Cell 77, 145-156) recently proposed that synthesis of the first phosphodiester bond of promoter-specific transcripts by RNA polymerase II is independent of ATP and general transcription factors TFIIE and TFIIH. Here we investigate this model. Taken together, our findings indicate that ATP, TFIIE, and TFIIH can have a profound effect on the efficiency of transcription initiation. First, we observe that synthesis of the first phosphodiester bond of transcripts initiated at the adenovirus 2 major late promoter depends strongly on ATP, TFIIE, and TFIIH in a transcription system reconstituted with RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and TFIIF. Second, we demonstrate that, in this enzyme system, ATP-dependent activation of transcription initiation can occur immediately prior to synthesis of the first phosphodiester bond of nascent transcripts. Finally, we demonstrate that the activated initiation complex is unstable and decays rapidly to an inactive state in the presence of the inhibitor ATP-gammaS (adenosine 5'-O-(thio)triphosphate), even during reiterative synthesis of abortive transcripts.
RNA聚合酶II合成启动子特异性转录本的前8至10个键时对ATP辅因子的需求已得到充分证实。然而，ATP是在新生转录本合成的第一个磷酸二酯键形成时就需要，还是在稍晚阶段才需要，仍存在争议。古德里奇和蒂坚（古德里奇，J.A.，和蒂坚，R.（1994年）《细胞》77卷，第145 - 156页）最近提出，RNA聚合酶II合成启动子特异性转录本的第一个磷酸二酯键与ATP以及通用转录因子TFIIE和TFIIH无关。在此我们对该模型进行研究。综合来看，我们的研究结果表明，ATP、TFIIE和TFIIH对转录起始效率可能有深远影响。首先，我们观察到在由RNA聚合酶II、TFIIH以及重组TBP、TFIIB、TFIIE和TFIIF重构的转录系统中，腺病毒2主要晚期启动子起始的转录本的第一个磷酸二酯键的合成强烈依赖于ATP、TFIIE和TFIIH。其次，我们证明，在这个酶系统中，转录起始的ATP依赖性激活可以在新生转录本的第一个磷酸二酯键合成之前立即发生。最后，我们证明，即使在流产转录本的反复合成过程中，在抑制剂ATP - γS（腺苷5'-O-(硫代)三磷酸）存在的情况下，活化的起始复合物也是不稳定的，会迅速衰变为无活性状态。
