Brezovská Barbora, Narasimhan Subhash, Šiková Michaela, Šanderová Hana, Kovaľ Tomáš, Borah Nabajyoti, Shoman Mahmoud, Pospíšilová Debora, Vaňková Hausnerová Viola, Tužinčin Dávid, Černý Martin, Komárek Jan, Janoušková Martina, Kambová Milada, Halada Petr, Křenková Alena, Hubálek Martin, Trundová Mária, Dohnálek Jan, Hnilicová Jarmila, Žídek Lukáš, Krásný Libor
Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czechia.
Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czechia.
J Bacteriol. 2024 Dec 19;206(12):e0006624. doi: 10.1128/jb.00066-24. Epub 2024 Nov 5.
In mycobacteria, σ is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of σ. The search revealed a number of proteins; prominent among them was MoaB2. The σ-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (, closely related σ) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σ was identified to play a role in the σ-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σ-dependent (but not σ-dependent) transcription and may increase the stability of σ in the cell. We propose that MoaB2, by sequestering σ, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σ, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σ, the primary sigma factor, and characterize its effects on transcription and σ stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.
在分枝杆菌中，σ是主要的σ因子。这种必需蛋白与RNA聚合酶（RNAP）结合，介导管家基因的转录起始。我们对分枝杆菌中该因子的了解有限。在此，我们对σ的相互作用伙伴进行了无偏向性搜索。搜索发现了许多蛋白质；其中突出的是MoaB2。通过多种方法验证并表征了σ与MoaB2的相互作用，结果表明这种相互作用可能不需要RNAP且具有特异性，因为替代σ因子（密切相关的σ）不与MoaB2相互作用。通过X射线晶体学解析了MoaB2的结构。通过免疫沉淀和核磁共振，确定了σ独特的、无结构的N端结构域在σ与MoaB2的相互作用中发挥作用。功能实验随后表明，MoaB2抑制σ依赖性（但不抑制σ依赖性）转录，并可能增加细胞中σ的稳定性。我们提出，MoaB2通过隔离σ，具有调节基因表达的潜力。总之，本研究发现了分枝杆菌σ的一种新的结合伙伴，为今后对这一现象的研究铺平了道路。重要性分枝杆菌可导致严重的人类疾病，如结核病和麻风病。分枝杆菌的转录机制独特，包含RbpA、CarD等转录因子以及与RNA聚合酶（RNAP）核心相互作用的小RNA Ms1。在此，我们通过鉴定MoaB2作为主要σ因子σ的相互作用伙伴，扩展了我们对分枝杆菌转录装置的认识，并表征了其对转录和σ稳定性的影响。这些信息扩展了我们对分枝杆菌RNAP亚基相互作用伙伴的认识，为未来抗分枝杆菌化合物的开发提供了机会。
