Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.
Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.
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DOI：
10.1073/pnas.2300282120
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发表时间：
2023-05-30
影响因子：
11.1
通讯作者：
Lin, Wei
中科院分区：
文献类型：
--
作者：
Shi, Jing;Feng, Zhenzhen;Xu, Juncao;Li, Fangfang;Zhang, Yuqiong;Wen, Aijia;Wang, Fulin;Song, Qian;Wang, Lu;Cui, Hong;Tong, Shujuan;Chen, Peiying;Zhu, Yejin;Zhao, Guoping;Wang, Shuang;Feng, Yu;Lin, Wei
关键词：
双组分信号转导系统通过组氨酸激酶传感器和反应调节器（RR）在细胞对复杂环境刺激的适应中发挥重要作用。在放线菌中，OmpR/PhoB亚家族蛋白质的孤儿RR称为GlnR，其全局协调涉及氮、碳和磷酸盐代谢的基因的转录。然而，其内在机制仍不清楚。在这里，使用晶体学，冷冻电子显微镜和生化分析，我们证明GlnR通过与σ区域4和RNAP β flap亚基合作与DNA元件相互作用来激活转录。我们还确定了以前未观察到的协作接口之间的四个GlnR的原体和启动子DNA或RNAP保守结构域。这些相互作用既保持了GlnR依赖性转录激活复合物（GlnR-TAC）的稳定性，又促进了有效的转录起始。在放线菌中，OmpR/PhoB亚家族的GlnR是一种孤儿反应调节蛋白，在放线菌中负责氮、碳和磷代谢的基因表达的全局协调。尽管许多研究者试图阐明GlnR依赖性转录激活的机制，但由于缺乏GlnR依赖性转录激活复合物（GlnR-TAC）的整体结构，进展受到阻碍。在这里，我们报告的C-末端的GlnR（GlnR_DBD）的DNA结合结构域的共晶体结构，在复杂的调节顺式元件的DNA和冷冻EM结构的GlnR-TAC，其中包括结核分枝杆菌RNA聚合酶，GlnR，和一个启动子含有四个良好的特点保守GlnR结合位点。这些结构说明了四个GlnR原聚体如何以头对尾的方式协调与启动子DNA结合，其中GlnR的四个N-末端受体结构域（GlnR-REC）桥接GlnR_DBD和RNAP核心酶。结构分析还揭示了GlnR-TAC通过GlnR与RNAP的保守β瓣、σ AR 4、αCTD和αNTD结构域之间的复杂蛋白质-蛋白质相互作用而稳定，这通过我们的生化测定进一步证实。总之，这些结果揭示了一个全球性的转录激活机制的主调节GlnR和其他OmpR/PhoB亚家族蛋白，并提出了一个独特的模式，细菌转录调控。
Two-component signal transduction systems play essential roles in cellular adaptation to complex environmental stimuli through a histidine kinase sensor and a response regulator (RR). In actinobacteria, an orphan RR of the OmpR/PhoB subfamily proteins called GlnR globally coordinates transcription of genes involved in nitrogen, carbon, and phosphate metabolisms. However, the underlying mechanism remains obscure. Here, using crystallography, cryo–electron microscopy, and biochemical assays, we demonstrate that GlnR activates transcription by interacting with DNA elements through cooperating with σ region 4 and RNAP β flap subunit. We also identify previously unobserved collaborative interfaces between four GlnR protomers and the promoter DNA or RNAP conserved domains. These interactions both retain the stability of the GlnR-dependent transcription activation complex (GlnR-TAC) and promote efficient transcription initiation. In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein–protein interactions between GlnR and the conserved β flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.
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影响因子：
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作者：
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