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。这一理解的进展受困于 可视化瞬变中间体，这些中间体是稳定状态之间关键转变的基础 转录循环，以及可视化复杂的大分子组装涉及的困难 管理，结构问题，其中X射线结晶学有严重的限制。 而稳定的RNAP状态在转录周期周围(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)相对 人们很好地描述和理解了稳定状态之间的转变，但对这些转变的理解却很少。 主要过渡包括： 全酶启动子dnaè开放启动子复合体(启动) 开放启动子复合体è延伸复合体(启动子逃逸，σ解离) 延长复合体è核心RNAPDNA完成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，这些中间产物非常 对结构生物学的挑战。 在转录周期的每个阶段，RNAP的功能都受到与外源基因的相互作用的调节 监管因素。含有外源基因的转录复合体的组装和结晶 监管机构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)现在提供了一种结构和 这些中间体和大型组件的机械表征。我们将使用低温电子 显微镜，结合X射线结晶学和其他方法来利用这一点 机会，并提供细菌转录周期的完整特征。

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. Advances in this understanding are stuck on the difficulty of visualizing transient intermediates that underlie the key transitions 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 è elongation complex (promoter escape, σ dissociation) Elongation complex è 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.