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: 10394344
• 负责人: Seth A. Darst
• 金额: $ 83.56万
• 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/10394344
• 关键词: Antibiotics; Bacteria; Bacterial RNA; Behavior; Binding; Biology; Chemicals; Combined Modality Therapy; Complex; Cryoelectron Microscopy; DNA; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Dissociation; Elements; Enzymes; Frequencies; Gene Expression; Genetic Transcription; Goals; Holoenzymes; Lead; Methods; Molecular Conformation; Molecular Machines; Prokaryotic Cells; RNA; Regulation; Rifampicin resistance; Rifampin; Series; Structure; Transcript; Tuberculosis; antimicrobial; inhibitor; insight; interest; man; melting; promoter; resistant strain; small molecule; structural biology; three dimensional structure; transcription termination

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. Moreover, a complete understanding of how a complex, molecular machine uses binding and chemical energy to effect conformational changes that drive the cycle, and how regulators modulate the cycle, is of fundamental interest. The transcription cycle is marked by a series of stable complexes (core è holo è RPo è EC) that interconvert through transient intermediates. The transitions between stable states are points of heavy regulation that are poorly understood due to the lack of structural information. Major transitions include: Holoenzyme + promoter DNA è open promoter complex (initiation) Open promoter complex è elongation complex (promoter escape, s dissociation) Elongation complex è core RNAP + DNA + completed RNA transcript (termination) Each of these transitions is characterized by unstable, transient intermediates that are extremely challenging for structural biology. Cryo-electron microscopy (cryo-EM) has emerged as a powerful method to visualize these transient states. We are combining cryo-EM with other approaches to mechanistically and structurally characterize transient intermediates that govern transitions in the bacterial transcription cycle, including promoter melting, the initiation to elongation transition, and transcription termination. These findings will provide insight into the behavior of macromolecular machines throughout biology.

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。此外，完全了解复杂的方式， 分子机使用结合和化学能来实现概念变化，以驱动驱动 周期以及监管机构如何调节周期，具有基本利益。 转录周期以一系列稳定的复合物（核心èloèrpoèEC）为标志 通过瞬态中间体互连。稳定状态之间的过渡是 由于缺乏结构信息而被理解的严重调节。主要过渡 包括： Holoenzyme +启动子DNAè开放式启动子络合物（启动） 开放式启动子复合伸长复合物（启动子逃生，分离） 伸长络合物è核RN​​AP + DNA +完成的RNA转录本（终止） 这些过渡中的每一个的特征是不稳定的瞬态中间体，这些中间体极为 对结构生物学的挑战。冷冻电子显微镜（Cryo-EM）已成为强大的 可视化这些瞬态状态的方法。我们正在将冷冻EM与其他方法相结合 机械和结构上表征了控制在 细菌转录周期，包括启动子熔化，伸长转变的主动性和 转录终止。这些发现将为大分子的行为提供洞察力 整个生物学的机器。