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: 9271202
• 负责人: Seth A. Darst
• 金额: $ 81.12万
• 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 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9271202
• 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; public health relevance; resistant strain; small molecule; structural biology; three dimensional structure

 DESCRIPTION (provided by applicant): 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 transition 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 > elongation complex (promoter escape, σ dissociation) Elongation complex > 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.

 描述（由申请人提供）：转录是基因表达的主要控制点，RNA聚合酶（RNAP）是转录的中心酶，从细菌到人都是保守的。我们的长期目标是了解转录机制及其调控。确定RNAP及其与DNA、RNA和调节因子的复合物的三维结构是重要的一步。我们专注于高度特征化的原核RNAP。通过对原核生物的研究，阐明了转录周期的基本要素，即起始、延伸和终止。对整个转录周期的详细结构和功能理解对于解释基因表达的基本控制和用小分子抗生素靶向RNAP是必不可少的。在这方面的理解的进展是停留在可视化的瞬态中间体，基础上的转录周期的稳定状态之间的关键转变的困难，以及可视化的复杂的大分子组装参与调控，结构问题，其中X射线晶体学有严重的局限性的困难。虽然转录循环周围的稳定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）相对较好地表征和理解，但稳定状态之间的转换知之甚少。主要过渡包括：全酶+启动子DNA →开放启动子复合体（起始）开放启动子复合体>延伸复合体（启动子逃逸，σ解离）延伸复合体>核心RNAP + DNA +完成的RNA转录物（终止）这些转变中的每一个都以不稳定的、短暂的中间体为特征，这些中间体对结构生物学极具挑战性。在转录周期的每一个阶段，RNAP的功能都是通过与外部调控因子的相互作用来调节的。组装和结晶含有外源性调节因子的转录复合物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）现在提供了一个途径，这些中间体和大型组件的结构和机械特性。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，利用这个机会，并提供一个完整的表征细菌转录周期。