Structure, function, and regulation 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，都是转录的核心酶。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。此外，一个复杂的， 分子机器使用结合能和化学能来影响构象变化， 周期，以及监管机构如何调节周期，是根本的利益。 转录周期以一系列稳定的复合物（core <$holo <$RPo <$EC）为标志， 通过瞬时中间体相互转化。稳定状态之间的转换是 由于缺乏结构性信息，人们对繁重的监管知之甚少。重大转变 包括以下步骤： 全酶+启动子DNA è开放启动子复合物（起始） 开放启动子复合体è延伸复合体（启动子逃逸，s解离） 延伸复合物<$core 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。冷冻电子显微镜（cryo-EM）已成为一种强大的 方法来可视化这些瞬态。我们将冷冻电镜与其他方法相结合， 机械和结构表征瞬态中间体，其控制 细菌转录周期，包括启动子解链、起始至延伸转变，以及 转录终止这些发现将有助于深入了解大分子的行为， 生物学中的机器

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.