Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes.
Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes.
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DOI：
10.1016/j.molcel.2021.07.025
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发表时间：
2021-09-02
期刊：
影响因子：
16
通讯作者：
Buratowski S
中科院分区：
文献类型：
--
作者：
Baek I;Friedman LJ;Gelles J;Buratowski S
由纯化因子重组的RNA聚合酶II （RNA Pol II）转录表明，起始前复合物（PICs）可以通过TATA盒中因子的顺序结合来组装。然而，这些基础转录反应通常独立于激活剂和共激活剂。为了在更现实的条件下研究PIC组装，我们使用单分子显微镜来观察核提取物中依赖活化剂的反应过程中的因子动力学。令人惊讶的是，RNA Pol II， TFIIF和TFIIE可以在加载到pic之前预先组装在增强子结合的激活剂上，并且多个RNA Pol II复合物可以同时结合以创建一个局部簇。与TFIIF和TFIIE不同，TFIIH的结合是单一的，依赖于基础启动子。激活因子的停留时间为秒级。相比之下，在没有三磷酸核苷酸的情况下，PICs可以持续几分钟，尽管TFIIE即使在TFIIH结合后仍然出乎意料地保持动态。我们的动力学测量导致了一个新的分支模型的活化剂依赖PIC装配。Baek等人的单分子显微镜实验表明，RNA聚合酶II和基础转录因子TFIIF和TFIIE预先组装在UAS/增强子结合的激活剂上，准备装载到起始复合物中，TFIIH位于核心启动子处。转录激活因子动态地增强因子招募，在UAS/增强子上形成一个局部聚合酶簇。
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can preassemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly. Single-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes, with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer.
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影响因子：
16.8
作者：
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通讯作者：
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DOI：
10.1126/science.1198830
发表时间：
2011-03-11
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
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作者：
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