Dvir A, Conaway R C, Conaway J W
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 1996 Sep 20;271(38):23352-6. doi: 10.1074/jbc.271.38.23352.
It is well established that TFIIH-dependent transcription by RNA polymerase II requires a hydrolyzable ATP cofactor for synthesis of the first phosphodiester bond of nascent transcripts. Whether an ATP cofactor is also required after initiation for escape of RNA polymerase II from the promoter has, however, been controversial. We have now addressed this question directly by investigating the ability of RNA polymerase II transcription complexes containing short, approximately 5-8-nucleotide transcripts synthesized in the presence of limiting nucleotides to escape the promoter in the absence of an ATP cofactor in a basal transcription system reconstituted with purified RNA polymerase II and general initiation factors. Depletion of ATP had a profound effect on the ability of initiated complexes to progress into the elongation phase: whereas in the presence of ATP, the majority of transcription complexes could be chased away from the promoter-proximal region, most complexes deprived of ATP catalyzed synthesis of only a few phosphodiester bonds and then ceased elongation after synthesizing transcripts less than 10-14 nucleotides in length. A significant fraction of these transcripts could be extended following addition of ATP, indicating that they were contained in arrested, but potentially active elongation complexes. Like the ATP-requiring step in initiation, ATP-dependent suppression of arrest by RNA polymerase II at promoter-proximal sites is inhibited by adenosine 5'-O-(thio)triphosphate. Transcription complexes containing transcripts longer than 9-10 nucleotides are insensitive to inhibition by ATPgammaS, indicating that susceptibility to ATP-sensitive arrest is a property of very early elongation complexes. Taken together, our findings reveal a novel role for an ATP cofactor in transcription by RNA polymerase II.
众所周知，RNA聚合酶II依赖TFIIH的转录需要一种可水解的ATP辅因子来合成新生转录本的第一个磷酸二酯键。然而，在起始之后RNA聚合酶II从启动子逃逸是否也需要ATP辅因子一直存在争议。我们现在通过研究在有限核苷酸存在下合成的含有短的、约5 - 8个核苷酸转录本的RNA聚合酶II转录复合物，在由纯化的RNA聚合酶II和一般起始因子重构的基础转录系统中，在没有ATP辅因子的情况下从启动子逃逸的能力，直接解决了这个问题。ATP的消耗对起始复合物进入延伸阶段的能力有深远影响：在有ATP的情况下，大多数转录复合物可以从启动子近端区域被追赶离开，而大多数缺乏ATP的复合物仅催化合成少数磷酸二酯键，然后在合成长度小于10 - 14个核苷酸的转录本后停止延伸。添加ATP后，这些转录本中有很大一部分可以延伸，表明它们包含在停滞但潜在活跃的延伸复合物中。与起始过程中需要ATP的步骤一样，RNA聚合酶II在启动子近端位点对停滞的ATP依赖性抑制被5'-O-(硫代)三磷酸腺苷抑制。含有长度超过9 - 10个核苷酸转录本的转录复合物对ATPγS的抑制不敏感，这表明对ATP敏感停滞的敏感性是非常早期延伸复合物的一个特性。综上所述，我们的发现揭示了ATP辅因子在RNA聚合酶II转录中的新作用。
