Vishwakarma Rishi Kishore, Marechal Nils, Morichaud Zakia, Blaise Mickaël, Margeat Emmanuel, Brodolin Konstantin
Institut de Recherche en Infectiologie de Montpellier, Univ Montpellier, CNRS, Montpellier 34293, France.
Centre de Biologie Structurale, Univ Montpellier, CNRS, INSERM, Montpellier 34090, France.
Nucleic Acids Res. 2025 Apr 10;53(7). doi: 10.1093/nar/gkaf272.
Initiation of transcription requires the formation of the "open" promoter complex (RPo). For this, the σ subunit of bacterial RNA polymerase (RNAP) binds to the nontemplate strand of the -10 element sequence of promoters and nucleates DNA unwinding. This is accompanied by a cascade of conformational changes on RNAP, the exact mechanics of which remains elusive. Here, using single-molecule Förster resonance energy transfer and cryo-electron microscopy, we explored the conformational landscape of RNAP from the human pathogen Mycobacterium tuberculosis upon binding to a single-stranded DNA (ssDNA) fragment that includes the -10 element sequence (-10 ssDNA). We found that like the transcription activator RNAP-binding protein A, -10 ssDNA induced σ subunit loading onto the DNA/RNA channels of RNAP. This triggered RNAP clamp closure and unswiveling that are required for RPo formation and RNA synthesis initiation. Our results reveal a mechanism of ssDNA-guided RNAP maturation and identify the σ subunit as a regulator of RNAP conformational dynamics.
转录起始需要形成“开放”启动子复合物（RPo）。为此，细菌RNA聚合酶（RNAP）的σ亚基与启动子-10元件序列的非模板链结合，并促使DNA解旋。这伴随着RNAP上一系列的构象变化，其确切机制仍不清楚。在这里，我们使用单分子Förster共振能量转移和冷冻电子显微镜，研究了人类病原体结核分枝杆菌的RNAP与包含-10元件序列（-10 ssDNA）的单链DNA（ssDNA）片段结合后的构象变化情况。我们发现，与转录激活因子RNAP结合蛋白A一样，-10 ssDNA诱导σ亚基加载到RNAP的DNA/RNA通道上。这触发了RPo形成和RNA合成起始所需的RNAP钳夹关闭和旋转解耦。我们的结果揭示了ssDNA引导的RNAP成熟机制，并确定σ亚基是RNAP构象动力学的调节因子。
