Department of Microbiology and Immunology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
Department of Microbiology and Immunology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, China; Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing, 210023, China.
Biochem Biophys Res Commun. 2021 Mar 19;545:98-104. doi: 10.1016/j.bbrc.2021.01.078. Epub 2021 Feb 3.
A large class of bacterial RNA polymerase (RNAP) from low-G + C-content Gram-positive bacterial strains, such as the major human pathogen Staphylococcus aureus, not only contain five conserved subunits (α, α, β, β' and ω), but also has a δ subunit. Despite being first identified as unique, Gram-positive specific component of RNAP apoenzyme more than 30 years ago and reported to be essential for transcription, the structural basis and molecular mechanism of δ subunit in the regulation of transcription remain poorly understood. Here, we performed structural analyses, site-directed mutagenesis and biochemical assays to uncover the interactions of S. aureus δ subunit with RNAP core enzyme and DNA towards the understanding of its role in transcription regulation. Microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA) of the wild-type and mutated S. aureus δ subunit revealed the N-terminal domain of δ subunit directly binds to the β' jaw of S. aureus RNAP (SauRNAP), identified the key amino acid residues (F58, D61, D65, R67 and W81) of δ subunit involving in the binding with SauRNAP core enzyme, and uncovered the δ subunit C-terminal domain interferes with the interaction between DNA and SauRNAP core enzyme, by which transcription is regulated. Our results provide an excellent starting point for understanding the unique regulatory role and physiological function of δ subunit on transcription regulation in Gram-positive bacteria.
一大类低 G+C 含量革兰氏阳性细菌菌株（如主要的人类病原体金黄色葡萄球菌）的 RNA 聚合酶（RNAP）不仅含有 5 个保守亚基（α、α'、β、β'和ω），还含有一个δ亚基。尽管该δ亚基在 30 多年前被首次鉴定为 RNA 聚合酶无酶复合物的独特、革兰氏阳性特异性成分，并被报道对转录是必需的，但它在转录调控中的结构基础和分子机制仍知之甚少。在这里，我们进行了结构分析、定点突变和生化测定，以揭示金黄色葡萄球菌δ亚基与 RNAP 核心酶和 DNA 的相互作用，从而了解其在转录调控中的作用。野生型和突变型金黄色葡萄球菌δ亚基的微量热泳动（MST）和电泳迁移率变动分析（EMSA）表明，δ亚基的 N 端结构域直接与金黄色葡萄球菌 RNAP（SauRNAP）的β'夹结合，确定了涉及与 SauRNAP 核心酶结合的δ亚基关键氨基酸残基（F58、D61、D65、R67 和 W81），并揭示了δ亚基 C 端结构域干扰 DNA 与 SauRNAP 核心酶之间的相互作用，从而调节转录。我们的研究结果为理解δ亚基在革兰氏阳性菌转录调控中的独特调控作用和生理功能提供了一个很好的起点。
