Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.
Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.
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单细菌转录起始复合物中的构象异质性和气泡动力学。
DOI：
10.1093/nar/gkx1146
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发表时间：
2018-01-25
影响因子：
14.9
通讯作者：
Kapanidis AN
中科院分区：
文献类型：
--
作者：
Duchi D;Gryte K;Robb NC;Morichaud Z;Sheppard C;Brodolin K;Wigneshweraraj S;Kapanidis AN
转录起始是所有生物基因调控的重要步骤。在细菌中，启动子DNA首先被RNA聚合酶（RNAP）识别，产生初始封闭复合物。该复合物随后发生构象变化，导致DNA链分离，形成转录泡和RNAP启动子开放复合物;然而，构象变化的系列和顺序以及影响它们的因素尚不清楚。为了解决构象景观和转录起始的转变，我们应用单分子Förster共振能量转移（smFRET）固定大肠杆菌转录开放复合物。我们的研究结果揭示了RNAP-DNA复合物中存在两种稳定状态，其中启动子DNA似乎采用封闭和部分开放的构象，我们观察到大规模的转换，其中转录泡在开放和封闭状态之间波动;这些转换，大致发生在0.1秒的时间尺度上，与以前观察到的毫秒时间尺度的动态扩散开放复合物。突变研究表明RNAP的σ70区域3.2显著影响气泡动力学。我们的研究结果对转录起始的许多步骤都有影响，并支持一个弯曲-负载-开放模型，用于解释开放复合物形成过程中导致气泡开放的过渡序列。
Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.
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