During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ⁷⁰–RNAP and the λP_R promoter. Cryo-EM snapshots revealed that 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 the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ⁷⁰ domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

在细菌 RNA 聚合酶 （RNAP） 形成具有转录能力的开放复合物 （RPo） 的过程中，瞬时中间体在克服限速步骤之前堆积起来。这些相互转换的实时结构描述不可用。为了解决这一差距，我们在这里使用时间分辨低温电子显微镜 （cryo-EM） 在将大肠杆菌^{σ 70-RNAP} 和 λP_R 启动子混合后 120 ms 或 500 ms 捕获四个填充的中间体。冷冻电镜快照显示，转录气泡的上游边缘迅速解开配对，然后将两个保守的非模板链（nt 链）碱基逐步插入 RNAP 口袋中。随着 nt 链“读出”的延伸，RNAP 夹闭合，从活性位点裂隙中排出抑制性 σ⁷⁰ 结构域。模板链在 120 ms 内完全不成对，但仍然是动态的，表明未知的构象变化在后续步骤中完成了 RPo 形成。鉴于这些事件可能描述了许多细菌启动子处的 DNA 开放，本研究提供了对 DNA 序列如何调节 RPo 形成步骤的见解。