Department of Chemistry and Biochemistry, University of Colorado, 215 UCB, Boulder, CO 80309-0215, USA.
J Mol Biol. 2010 Mar 19;397(1):57-68. doi: 10.1016/j.jmb.2010.01.025. Epub 2010 Jan 18.
Transcription of mRNA genes requires that RNA polymerase II (Pol II) and the general transcription factors assemble on promoter DNA to form an organized complex capable of initiating transcription. Biochemical studies have shown that Pol II and TFIID (transcription factor IID) contact overlapping regions of the promoter, leading to the question of how these large factors reconcile their promoter interactions during complex assembly. To investigate how the TAF (TATA-binding protein-associated factor) subunits of TFIID alter the kinetic mechanism by which complexes assemble on promoters, we used a highly purified human transcription system. We found that TAFs sharply decrease the rate at which Pol II, TFIIB, and TFIIF assemble on promoter-bound TFIID-TFIIA. Interestingly, the slow step in this process is not recruitment of these factors to the DNA, but rather a postrecruitment isomerization of protein-DNA contacts that occurs throughout the core promoter. Our findings support a model in which Pol II and the general transcription factors rapidly bind promoter-bound TFIID-TFIIA, after which complexes undergo a slow isomerization in which the TAFs reorganize their contacts with the promoter to allow Pol II to properly engage the DNA. In this manner, TAFs kinetically repress basal transcription.
mRNA 基因的转录需要 RNA 聚合酶 II（Pol II）和一般转录因子组装在启动子 DNA 上，形成能够起始转录的有组织的复合物。生化研究表明，Pol II 和 TFIID（转录因子IID）接触启动子的重叠区域，这导致了一个问题，即这些大型因子如何在复合物组装过程中协调它们的启动子相互作用。为了研究 TFIID 的 TAF（TATA 结合蛋白相关因子）亚基如何改变复合物在启动子上组装的动力学机制，我们使用了高度纯化的人类转录系统。我们发现 TAFs 显著降低了 Pol II、TFIIB 和 TFIIF 在结合 TFIID-TFIIA 的启动子上组装的速率。有趣的是，这个过程中的缓慢步骤不是这些因子被招募到 DNA 上，而是在整个核心启动子中发生的蛋白质-DNA 接触的后续募集异构化。我们的发现支持这样一种模型，即 Pol II 和一般转录因子在结合 TFIID-TFIIA 的启动子上快速结合，然后复合物经历一个缓慢的异构化过程，其中 TAFs 重新组织它们与启动子的接触，以允许 Pol II 正确地与 DNA 结合。通过这种方式，TAFs 在动力学上抑制基础转录。
