Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom.
Waksman Institute and Department of Chemistry, Rutgers University, Piscataway, United States.
Elife. 2021 Oct 11;10:e70090. doi: 10.7554/eLife.70090.
Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPo). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyse RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock', model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.
转录起始是由 RNA 聚合酶 (RNAP) 解开启动子 DNA 开始的，形成具有催化能力的 RNAP-启动子复合物 (RPo)。尽管进行了广泛的研究，但启动子解开的机制仍不清楚，部分原因是中间产物在形成 RPo 的过程中具有瞬时性。在这里，我们使用单分子解开诱导的荧光增强来监测启动子解开，使用单分子荧光共振能量转移来监测 RNAP 夹的构象，分析了在一个共识的细菌核心启动子上形成 RPo 的过程。我们发现，RNAP 夹在启动子结合时是闭合的，在启动子解开过程中保持闭合，然后进一步闭合，将解开的 DNA 锁定在 RNAP 的活性中心裂缝中。我们的工作为在共识细菌启动子上解开启动子时发生的一系列构象变化定义了一个新的“结合-解开-加载-锁定”模型，并提供了在其他生物体和其他启动子上研究该过程所需的工具。
