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: 9071516
• 负责人: Seth A. Darst
• 金额: $ 79.1万
• 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/9071516
• 关键词: Antibiotics; Bacteria; Bacterial RNA; Catalytic Domain; Combined Modality Therapy; Complex; Cryoelectron Microscopy; DNA; DNA-Directed RNA Polymerase; Development; Dissociation; Elements; Enzymes; Frequencies; Gene Expression; Genetic Transcription; Goals; Holoenzymes; Lead; Prokaryotic Cells; RNA; Regulation; Rifampicin resistance; Rifampin; Route; Staging; Structure; Transcript; Tuberculosis; X-Ray Crystallography; antimicrobial; inhibitor/antagonist; insight; macromolecular assembly; man; promoter; public health relevance; 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(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，从细菌到人类都是保守的。我们的长期目标是了解转录的机制及其调控。确定RNAP及其与DNA、RNA和调节因子的复合体的三维结构是必不可少的一步。我们专注于高度特征化的原核生物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的了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是至关重要的。这方面的进展停留在可视化瞬时中间体的困难上，瞬时中间体是转录周期稳定状态之间的关键转变，以及可视化参与调控的复杂大分子组件的困难，X射线结晶学在这些结构问题上具有严重限制。虽然对转录循环周围稳定的RNAP状态(RNAP催化核心、RNAP全酶、RNAP全酶开放启动子复合体、RNAP延伸复合体)的描述和理解相对较好，但对稳定状态之间的转换了解较少。主要的转变包括：全酶+启动子开放启动子复合体(起始)开放启动子复合体&GT；延伸复合体(启动子逃逸，σ解离)延伸复合体&GT；核心RNAP+DNA+完整转录本(终止)每个转变的特征是不稳定的、瞬时的中间体，这对结构生物学来说是极具挑战性的。在转录周期的每个阶段，RNAP的功能都受到与外部调节因子的相互作用的调节。组装和结晶含有外部调控因子的转录复合体也给结构生物学带来了挑战。由于最近的进展，低温电子显微镜(CRYO-EM)现在提供了一种对这些中间体和大组件进行结构和机械表征的途径。我们将使用冷冻电子显微镜，结合X射线结晶学和其他方法，来利用这个机会，并提供细菌转录周期的完整特征。