TFIID Enables RNA Polymerase II Promoter-Proximal Pausing
TFIID Enables RNA Polymerase II Promoter-Proximal Pausing
复制标题
DOI：
10.1016/j.molcel.2020.03.008
复制
发表时间：
2020-05-21
期刊：
影响因子：
16
通讯作者：
Taatjes, Dylan J.
中科院分区：
文献类型：
--
作者：
Fant, Charli B.;Levandowski, Cecilia B.;Taatjes, Dylan J.
RNA聚合酶II（RNAPII）转录由前起始复合物（PIC）控制，所述前起始复合物包含TFIIA、TFIIB、TFIID、TFIIE、TFIIF、TFIIH、RNAPII和介体。启动后，RNAPII酶在转录不到100个碱基后暂停;确切地说，RNAPII暂停是如何执行和调节的仍然不清楚。为了解决具体的机制问题，我们重建人RNAPII启动子近端暂停在体外，完全与纯化的因素（无提取物）。正如预期的那样，NELF和DSIF增加暂停，P-TEFb促进暂停释放。出乎意料的是，PIC单独足以重建暂停，这表明RNAPII暂停是一种固有的PIC功能。与此一致，在用TATA结合蛋白（TBP）替换TFIID复合物后，暂停消失，并且PRO-seq实验揭示了在人或果蝇细胞中TFIID亚基急性耗尽（t = 60 min）后RNAPII暂停的广泛破坏。这些结果建立了RNAPII暂停的TFIID要求，并表明暂停调节因子可能直接或间接通过TFIID发挥作用。
RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.