Saecker Ruth M, Mueller Andreas U, Malone Brandon, Chen James, Budell William C, Dandey Venkata P, Maruthi Kashyap, Mendez Joshua H, Molina Nina, Eng Edward T, Yen Laura Y, Potter Clinton S, Carragher Bridget, Darst Seth A
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065 USA.
The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY USA.
bioRxiv. 2024 Mar 14:2024.03.13.584744. doi: 10.1101/2024.03.13.584744.
During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing σ-RNAP and the λP promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory σ domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation.
在细菌RNA聚合酶（RNAP）形成具有转录活性的开放复合物（RPo）的过程中，瞬时中间体在克服限速步骤之前会不断积累。目前尚无法实时获取这些相互转化的结构描述。为了填补这一空白，我们使用了时间分辨冷冻电子显微镜（cryo-EM）来捕获在混合σ-RNAP和λP启动子后120毫秒或500毫秒时出现的四种中间体。冷冻电镜快照显示，转录泡的上游边缘迅速解链，随后两个保守的非模板链（nt链）碱基逐步插入RNAP口袋。随着nt链“读出”的延伸，RNAP夹子关闭，将一个抑制性的σ结构域从活性位点裂隙中排出。模板链在120毫秒时完全解链，但仍保持动态，这表明在后续步骤中加载它的构象变化尚不清楚。由于这些事件可能描述了许多细菌启动子处的DNA解旋，这项研究为DNA序列如何调节RPo形成步骤提供了必要的见解。
