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.
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 聚合酶 II (Pol II)-TFIIF 和 Pol II-介体复合物：主要的稳定 Pol II 复合物及其在转录起始和重新起始中的活性。
DOI：
10.1128/mcb.24.4.1709-1720.2004
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发表时间：
2004
影响因子：
5.3
通讯作者：
Hahn,Steven
中科院分区：
文献类型：
--
作者：
Rani,PGeetha;Ranish,JeffreyA;Hahn,Steven
蛋白质纯化和消耗的研究被用来确定主要的稳定形式的RNA聚合酶II（Pol II）复合物中发现酵母grainaenuclear提取物。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。未观察到Pol II-Med-TFIIF复合物。Pol II和纯化的Pol II复合物在转录起始和再起始中的活性通过用纯化的Pol II或Pol II复合物补充耗尽总Pol II或总TFIIF的提取物来研究。我们发现，所有三种形式的Pol II可以补充Pol II耗尽提取物的转录起始，但Pol II-TFIIF具有最高的比活性。类似地，对于TFIIF转录活性的互补，Pol II-TFIIF具有比TFIIF高得多的比活性。虽然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在提取物中是动态的，其中单个聚合酶能够交换结合的介体和TFIIF。使用纯化的系统来检查转录再起始，我们发现Pol II-TFIIF在促进多轮转录中是活跃的，而Pol II-Med几乎是无活性的。这些结果表明，Pol II-Med和Pol II-TFIIF复合物都可以被募集用于转录起始，但只有Pol II-TFIIF复合物才能胜任转录再起始。
Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found inSaccharomyces cerevisiaenuclear 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.