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首页> 外文期刊>Molecular and Cellular Biology >RNA Polymerase II (Pol II)-TFIIF and Pol II-Mediator Complexes: the Major Stable Pol II Complexes and Their Activity in Transcription Initiation and Reinitiation
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RNA Polymerase II (Pol II)-TFIIF and Pol II-Mediator Complexes: the Major Stable Pol II Complexes and Their Activity in Transcription Initiation and Reinitiation

机译：RNA聚合酶II（Pol II）-TFIIF和Pol II-介体复合物：主要稳定的Pol II复合物及其在转录起始和重新启动中的活性

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Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts. About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med). No Pol II-Med-TFIIF complex was observed. The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes. We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity. Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity. Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF. Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive. These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation.
机译：蛋白质纯化和耗竭研究用于确定在酿酒酵母核提取物中发现的RNA聚合酶II（Pol II）复合物的主要稳定形式。发现约50％的Pol II与一般转录因子TFIIF（Pol II-TFIIF）相关，约20％的Pol II与介体（Pol-Med）相关。没有观察到Pol II-Med-TFIIF复合物。 Pol II和纯化的Pol II复合物在转录起始和重新启动过程中的活性通过用纯化的Pol II或Pol II复合物补充耗尽了总Pol II或总TFIIF的提取物来进行研究。我们发现，Pol II的所有三种形式都可以补充Pol II耗竭的提取物以进行转录起始，但是Pol II-TFIIF具有最高的比活性。同样，Pol II-TFIIF具有比TFIIF更高的比活性，可以互补TFIIF转录活性。尽管Pol II-TFIIF和Pol II-Med复合物在纯化时是稳定的，但我们发现这些复合物在转录条件下的提取物中是动态的，具有能够交换结合的介体和TFIIF的单一聚合酶。使用纯化的系统检查转录重新初始化，我们发现Pol II-TFIIF在促进多轮转录方面具有活性，而Pol II-Med几乎不起作用。这些结果表明，可以招募Pol II-Med和Pol II-TFIIF复合体进行转录起始，但是只有Pol II-TFIIF复合体才能够进行转录重新起始。

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