Transcription initiation by human RNA polymerase II visualized at single-molecule resolution
Transcription initiation by human RNA polymerase II visualized at single-molecule resolution
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DOI：
10.1101/gad.194936.112
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发表时间：
2012-08-01
影响因子：
10.5
通讯作者：
Tjian, Robert
中科院分区：
文献类型：
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作者：
Revyakin, Andrey;Zhang, Zhengjian;Tjian, Robert
四十年的经典生化分析已经确定了参与真核 RNA 聚合酶 II (Pol II) 转录起始的分子参与者，并在很大程度上确定了它们的功能。然而，Pol II 转录起始的动态图景及其调控机制的理解仍然难以捉摸，部分原因是传统整体生物化学的固有局限性。在这里，我们开始使用荧光视频显微镜以单分子分辨率剖析由重建的人类 Pol II 系统引导的启动子特异性转录起始。我们从单个 DNA 模板中检测了几轮随机的人类 Pol II 转录，观察到启动子突变引起的转录减弱，观察到激活子 Sp1 引起的转录增强，并将转录信号与单个 DNA 模板上的 Holo-TFIID 分子的实时相互作用相关联。这种集成的单分子方法应该适用于研究其他复杂的生物过程。
Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.