Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Nucleic Acids Res. 2019 Jul 26;47(13):7094-7104. doi: 10.1093/nar/gkz470.
Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.
细菌 RNA 聚合酶（RNAP）与细胞外功能（ECF）σ因子形成不同的全酶，以启动特定的基因表达程序。在这项研究中，我们报告了大肠杆菌转录起始复合物的冷冻电镜结构，该复合物包含最受研究的细菌 ECF σ因子 σE（Ec σE-RPo），以及 3.1 Å 的结核分枝杆菌转录起始复合物的晶体结构，其中含有嵌合 σH/E（Mtb σH/E-RPo）。Ec σE-RPo 的结构揭示了组装大肠杆菌 σE-RNAP 全酶以及大肠杆菌 σE 识别和展开启动子所必需的关键相互作用。此外，这两个结构都表明，两个 ECF σ 因子的非保守连接子（σ2/σ4 连接子）插入到活性中心裂隙中，并通过 RNA 出口通道离开。我们对 27670 个 ECF σ 因子进行了二级结构预测，发现它们的非保守连接子可能以类似的方式进入和离开 RNAP 活性中心裂隙。进一步的生化结果表明，这种 σ2/σ4 连接子在 ECF σ 因子介导的转录起始过程中的 RPo 形成、无效产物和启动子逃避中起着重要作用。
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