Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN 55912, USA.
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China.
Nucleic Acids Res. 2024 Feb 9;52(3):1471-1482. doi: 10.1093/nar/gkad1231.
Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.
转录激活是转录起始过程中调控的关键步骤，也是响应不同刺激和应激因素的经典检查点。大肠杆菌 NarL 是一种硝酸盐响应的全局转录因子，控制着近 100 个基因的表达。然而，NarL 介导的转录激活的分子机制尚未完全明确。本研究以 yeaR 启动子为模型，在 3.2 Å分辨率下解析了 NarL 依赖性转录激活复合物（TAC）的冷冻电镜结构。该结构显示，NarL 二聚体与启动子 DNA 的-43.5 位点结合，其 C 端结构域（CTD）不仅与 DNA 结合，还与 RNA 聚合酶亚基α CTD（αCTD）相互作用。体内和体外转录实验进一步证实了这些 NarL 介导的相互作用在转录激活中的关键作用。此外，NarL 二聚体与 DNA 的结合方式与 II 类 TAC 结构中观察到的不同。与典型的 II 类激活机制不同，NarL 不与 σ4 相互作用，而 RNAP αCTD 结合在 NarL 相反的 DNA 结合面上。本研究结果为 yeaR 启动子上 NarL 依赖性转录激活的详细机制理解提供了结构基础，并揭示了一种潜在的转录激活新机制。
