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（由适用提供）：转录是从细菌到人组成的基因表达和RNA聚合酶（RNAP）的主要控制点，是转录的中心酶。我们的长期目标是了解转录的机制及其调节。确定RNAP的三维结构及其与DNA，RNA和调节因子的复合物是重要的一步。我们专注于高度特征的原核生物RNAP。转录周期，倡议，伸长和终止的基本要素通过原核生物的研究阐明。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至关重要。这种理解的进步困难地可视化瞬态中间体的困难，这些中间体是转录周期稳定状态之间的关键过渡的基础，以及可视化参与调节的复杂的大分子组件的困难，X射线晶体学具有严重限制的结构问题。虽然稳定的RNAP状态围绕转录周期（RNAP催化核心，RNAP Holoenzyme，RNAP Holoenzyme开放启动子络合物复合物，RNAP伸长复合物）的表征相对较好，但稳定状态之间的过渡却相对良好。主要过渡包括：Holoenzyme +启动子DNAè开放启动子络合物（启动）开放启动子络合物>伸长式复合物（启动子逃脱，σ离解）伸长率>核心RNAP + DNA +完成的RNA转录（终止）这些过渡都由各个稳定的瞬时中间体表征，这些过渡者具有极具挑战性的结构性挑战性的挑战。在转录周期的每个阶段，RNAP函数都由与外部调节因子的相互作用调节。包含外在调节剂的组装和结晶转录复合物也对结构生物学提出了挑战。由于最近的进步，冷冻电子显微镜（Cryo-EM）现在为这些中间体和大型组件的结构和机械表征提供了途径。我们将使用冷冻电子显微镜与X射线晶体学和其他方法结合使用，以利用这一机会并提供细菌转录周期的完整表征。