Structure, function, and regulation of the bacterial transcription cycle

项目摘要 转录是基因表达的主要控制点，RNA聚合酶(RNAP)，保守 从细菌到人类，都是转录的中心酶。我们的长期目标是了解 转录机制及其调控。确定RNAP和RNAP的三维结构 它与DNA、RNA和调节因子的复合体，是必不可少的一步。我们高度关注 鉴定了原核生物的RNAP。 阐明了转录周期的基本要素，即起始、延伸和终止 通过对原核生物的研究。对整个的结构和功能有详细的了解 转录循环是解释基因表达的基本控制和靶向的关键 RNAP加小分子抗生素。此外，完全理解一个复杂的， 分子机器使用结合和化学能来影响构象变化，从而驱动 周期，以及监管者如何调节周期，关系到根本利益。 转录周期由一系列稳定的复合体(coreèholoèRPOèEC)标记 通过瞬时中间体进行相互转化。稳定状态之间的跃迁是 由于缺乏结构性信息，人们对严格的监管知之甚少。重大转型 包括： 全酶启动子DNAèOpen启动子复合体(启动) 开放启动子复合体è延伸复合体(启动子逃逸，S解离) 延伸复合体ècore RNAP DNA完成的RNA转录本(终止) 这些转变中的每一个都具有不稳定的、瞬时的中间产物，这些中间产物极其 对结构生物学的挑战。低温电子显微镜(Cryo-EM)已经成为一种强大的 方法来可视化这些瞬变状态。我们正在将冷冻-EM与其他方法相结合 从机械和结构上表征控制过渡过程的过渡中间体 细菌转录循环，包括启动子融化、从起始到延伸的转变，以及 转录终止。这些发现将为深入了解大分子的行为提供帮助。 整个生物学中的机器。

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.