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 转录是基因表达和RNA聚合酶（RNAP）的主要控制点， 从细菌到人类，都是转录的中心酶。我们的长期目标是了解 转录机制及其调控。确定RNAP的三维结构， 其与DNA、RNA和调节因子的复合物是重要的步骤。我们专注于 特征的原核RNAP。 阐明了转录周期的基本要素，起始、延伸和终止 通过研究原核生物。详细了解整个系统的结构和功能 转录周期对于解释基因表达的基本控制和靶向 RNAP与小分子抗生素。这种理解的进展被困在以下困难上： 可视化瞬态中间体，这些中间体是稳定状态之间的关键转变的基础， 转录周期，以及可视化复杂的大分子组装涉及的困难， 调节，结构问题，其中X射线晶体学具有严重的局限性。 而转录周期周围的稳定RNAP状态（RNAP催化核心，RNAP 全酶、RNAP全酶开放启动子复合物、RNAP延伸复合物）相对 虽然已经被很好地描述和理解，但是对稳定状态之间的转变却知之甚少。 主要过渡包括： 全酶+启动子DNA开放启动子复合物（起始） 开放启动子复合体<$延伸复合体（启动子逃逸，σ解离） 延伸复合物<$cRNA核心RNAP + DNA +完成的RNA转录物（终止） 每一个过渡的特点是不稳定的，短暂的中间体， 结构生物学的挑战。 在转录周期的每一个阶段，RNAP的功能都是通过与外源基因的相互作用来调节的。 调节因素。组装和结晶含有外源性的转录复合物 调控因子也对结构生物学提出了挑战。 由于最近的进展，低温电子显微镜（cryo-EM）现在提供了一种结构和 这些中间体和大组件的机械表征。我们将使用低温电子 显微镜，结合X射线晶体学和其他方法，来利用这一点。 的机会，并提供了细菌转录周期的完整表征。