Structure, function, and regulation of the bacterial transcription cycle

细菌转录周期的结构、功能和调控

• 批准号: 10388954
• 负责人: Seth A. Darst
• 金额: $ 5.36万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388954
• 关键词: Antibiotics; Bacteria; Bacterial RNA; Catalytic Domain; Combined Modality Therapy; Complex; Cryoelectron Microscopy; Crystallization; DNA; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Dissociation; Elements; Enzymes; Frequencies; Gene Expression; Genetic Transcription; Goals; Holoenzymes; Lead; Macromolecular Complexes; Prokaryotic Cells; RNA; Regulation; Rifampicin resistance; Rifampin; Route; Structure; Transcript; Tuberculosis; X-Ray Crystallography; antimicrobial; inhibitor/antagonist; insight; macromolecular assembly; man; promoter; resistant strain; small molecule; structural biology; three dimensional structure

Project Summary Transcription is the major control point of gene expression and RNA polymerase (RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. The basic elements of the transcription cycle, initiation, elongation, and termination, were elucidated through study of prokaryotes. A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics. Advances in this understanding are stuck on the difficulty of visualizing transient intermediates that underlie the key transitions between stable states of the transcription cycle, and the difficulty of visualizing complex macromolecular assemblies involved in regulation, structural problems where X-ray crystallography has severe limitations. While the stable RNAP states around the transcription cycle (RNAP catalytic core, RNAP holoenzyme, RNAP holoenzyme open promoter complex, RNAP elongation complex) are relatively well characterized and understood, the transitions between the stable states are poorly understood. Major transitions include: Holoenzyme + promoter DNA è open promoter complex (initiation) Open promoter complex è elongation complex (promoter escape, σ dissociation) Elongation complex è core RNAP + DNA + completed RNA transcript (termination) Each of these transitions are characterized by unstable, transient intermediates that are extremely challenging for structural biology. At every stage of the transcription cycle, RNAP function is modulated by interactions with extrinsic regulatory factors. Assembling and crystallizing transcription complexes containing extrinsic regulators also presents challenges for structural biology. Due to recent advances, cryo-electron microscopy (cryo-EM) now offers a route to structural and mechanistic characterization of these intermediates and large assemblies. We will use cryo-electron microscopy, in combination with X-ray crystallography and other approaches, to exploit this opportunity and provide a complete characterization of the bacterial transcription cycle.

项目摘要 转录是构成基因表达和RNA聚合酶（RNAP）的主要控制点 从细菌到人，是转录的中心酶。我们的长期目标是了解 转录机理及其调节。确定RNAP的三维结构 它与DNA，RNA和调节因子的复合物是重要的一步。我们专注于高度关注 特征是原核生物RNAP。 阐明了转录周期，主动性，伸长和终止的基本要素，阐明了 通过研究原核生物。对整个的详细结构和功能理解 转录周期对于解释基因表达的基本控制和目标至关重要 带有小分子抗生素的RNAP。这种理解的进步困扰 可视化瞬态中间体，这些中间位于稳定状态之间的关键过渡基础 转录周期以及可视化复合物大分子组件的困难 X射线晶体学具有严重局限性的调节，结构性问题。 而稳定的RNAP在转录周期（RNAP催化核心，RNAP）周围表示 Holoenzyme，RNAP Holoenzyme开放启动子络合物，RNAP伸长络合物相对较 良好的特征和理解，稳定状态之间的过渡知之甚少。 主要过渡包括： Holoenzyme +启动子DNAè开放启动子络合物（启动） 开放式启动子复合物延伸复合物（启动子逃脱，σ离解） 伸长络合物}核心RNAP + DNA +完成的RNA转录（终止） 这些转变中的每一个都以不稳定的瞬态中间体为特征 对结构生物学的挑战。 在转录周期的每个阶段，RNAP函数都通过与外部的相互作用来调节 监管因素。组装和结晶的转录复合物，含有外部 监管机构还提出了结构生物学的挑战。 由于最近的进步，冷冻电子显微镜（Cryo-EM）现在提供了结构性和 这些中间体和大型组合的机械表征。我们将使用冷冻电子 显微镜，结合X射线晶体学和其他方法，以利用这一点 机会并提供细菌转录周期的完整表征。