Structural study of direct associations between cellular RNA polymerase and regulatory factors during the transcription cycle

转录周期中细胞 RNA 聚合酶与调节因子之间直接关联的结构研究

• 批准号: 10609003
• 负责人: Katsuhiko Murakami
• 金额: $ 38.8万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609003
• 关键词: Archaea; Archaeal RNA; Bacteria; Bacterial RNA; Biochemical; Cell physiology; Cells; Cellular biology; Complex; Coupled; Cryoelectron Microscopy; Crystallization; DNA Repair; DNA-Directed RNA Polymerase; Development; Disease; Enzymes; Escherichia coli; Gene Expression; Genetic Transcription; Goals; Holoenzymes; Human; Life; Ligands; Macromolecular Complexes; Mainstreaming; Maintenance; Methods; Molecular Structure; Motor; Nature; Pathway interactions; Play; Polymerase; Research; Resolution; Role; Structure; Transcription Elongation; Transcription Initiation; Transcription Process; Transcriptional Regulation; Visualization; X-Ray Crystallography; drug discovery; improved; insight; macromolecular assembly; prevent; promoter; response; structural biology; termination factor; transcription termination

Project Summary The long-term goal of our research is to understand transcription mechanisms of cellular RNA polymerase. Our research has made major contributions to provide structures of RNA polymerase at the different stage of transcription cycle. During the next five years, we will continue to study the transcription machinery in bacteria and archaea to provide insights of the fundamental mechanism of transcription, which is conserved from bacteria to human. Over the last 20 years, X-ray crystallography has been playing a major role in the structural biology of RNA polymerase and revealed many important structures. RNA polymerases at evident stages in the mainstream of the transcription cycle are often referred to RNA polymerase core enzyme, holoenzymes, open complex with a strong promoter, transcription initiation and elongation complexes. These structures have been well characterized due to their stable natures. A challenge in the structural biology of cellular RNA polymerase is to visualize transient interactions of RNA polymerase with other regulatory factors and ligands that occur in between each evident stage in the mainstream or at the branched pathways from the mainstream of transcription cycle. Due to their elusive natures, crystallization of such macromolecular assemblies has been a bottleneck and thus limited the approach by X-ray crystallography. In the last couple of years, the resolution of macromolecular structures determined by cryo-electron microscopy (cryo-EM) has been drastically improved due to multiple technical advances, allowing us to visualize heterogenous and large assembly of macromolecular complexes. We will use structural biology methods including both cryo-EM and X-ray crystallography together with other biochemical approaches to visualize these transient interactions and investigate their effects for transcription process. Major targets of our study are: 1) the interaction between the Escherichia coli RNA polymerase and DksA/ppGpp for transcription regulation during the stringent response; 2) the interaction between bacterial RNA polymerase and ATP-dependent motor enzyme for rescuing stalled RNA polymerase at the end of transcription cycle; and 3) the interaction between archaeal RNA polymerase and ATP-dependent transcription termination factor Eta for disrupting a stalled transcription elongation complex to initiate the transcription-coupled DNA repair.

项目摘要 我们的长期研究目标是了解细胞RNA聚合酶的转录机制。 我们的研究为提供RNA聚合酶不同阶段的结构做出了重大贡献 转录周期。在接下来的五年里，我们将继续研究转录机制， 细菌和古细菌提供的转录的基本机制，这是保守的见解 从细菌到人类。 在过去的20年里，X射线晶体学在RNA的结构生物学中发挥了重要作用 聚合酶，并揭示了许多重要的结构。RNA聚合酶在主流中处于明显阶段 转录循环的核心酶通常被称为RNA聚合酶、全酶、开放复合物 具有强启动子、转录起始和延伸复合物。这些结构已经很好地 其特征在于其稳定的性质。细胞RNA聚合酶的结构生物学中的一个挑战是 为了可视化RNA聚合酶与其他调节因子和配体的瞬时相互作用， 在主流中的每个明显阶段之间或在来自主流的分支路径处， 转录周期由于其难以捉摸的性质，这种大分子组装体的结晶已经 一直是一个瓶颈，因此限制了X射线晶体学的方法。 近几年来，低温电子学对大分子结构的分辨 由于多种技术进步，冷冻电镜（cryo-EM）得到了极大的改进，使我们能够 可视化大分子复合物的异质和大型组装。我们将使用结构生物学 方法包括冷冻EM和X射线晶体学以及其他生物化学方法， 可视化这些瞬时相互作用并研究它们对转录过程的影响。主要目标 我们的研究内容是：1）大肠杆菌RNA聚合酶与DksA/ppGpp的相互作用， 严格反应过程中的转录调控; 2）细菌RNA聚合酶之间的相互作用 和ATP依赖性马达酶，用于在转录周期结束时拯救停滞的RNA聚合酶; 古细菌RNA聚合酶与ATP依赖的转录终止的相互作用 用于破坏停滞的转录延伸复合物以启动转录偶联DNA的因子Eta 修复.