Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, 16802, USA.
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia.
Nat Commun. 2021 Jan 22;12(1):528. doi: 10.1038/s41467-020-20776-y.
Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β' lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.
核糖体 RNA (rRNA) 在快速生长的细菌中表达水平最高，在应激条件下，全局转录调节因子 DksA 和魔斑 pppGpp 会使 rRNA 大幅下调。在此，我们利用冷冻电镜技术确定了带有和不带有 DksA/ppGpp 的大肠杆菌 RNA 聚合酶 (RNAP) σ 全酶在 rRNA 启动子识别过程中的结构。RNAP 通过二聚化的α亚基羧基末端结构域与 UP 元件结合，并利用 σ 指和β'盖将模板 DNA 扭结，以选择有利于快速启动子逃避的转录起始位点。启动子结合诱导σ结构域 2 的构象变化，打开 DNA 加载门，并将 σ 从 RNAP 裂隙中逐出，以促进开放复合物的形成。DksA/ppGpp 的结合也打开了 DNA 加载门，但其与 σ 逐出不偶联，并阻碍了开放复合物的形成。这些结果为 rRNA 转录的异常活跃及其对 DksA/ppGpp 的敏感性提供了分子基础。
