Structural Studies of RNA Polymerase II Transcription Initiation and Elongation

RNA 聚合酶 II 转录起始和延伸的结构研究

基本信息

批准号：
10596100
负责人：
Guillermo Alberto Calero
金额：
$ 31.8万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10596100
关键词：
Active Sites Binding Biology Catalysis Chemistry Chromatin Complex Coupled Cryoelectron Microscopy Crystallization Crystallography Custom DNA DNA Damage DNA Polymerase I DNA Polymerase III DNA lesion DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase Data Data Collection Data Set Electron Microscopy Elements Elongation Factor Enzymes Eukaryota Event Exposure to Foundations Genes Genetic Transcription Hydrolysis In Vitro Individual Ions Knowledge Lasers Life Mediating Messenger RNA Metals Methodology Modeling Molecular Molecular Conformation Molecular Evolution Motion Nature Nucleotides Organism Peptide Initiation Factors Physiologic pulse Play Polymerase Positioning Attribute Process Publishing RNA RNA Polymerase II RNA chemical synthesis Radiation induced damage Reaction Regulation Resolution Roentgen Rays Role Seminal Site Sodium Chloride Specific qualifier value Structure System Technology Temperature Time Transcription Elongation Transcription Initiation Transcription Process Visualization Work X-Ray Crystallography beamline chemical group electron density experimental study free-electron laser insight millisecond movie mutant particle scaffold technological innovation technology development time interval time use transcription factor

项目摘要

PROJECT SUMMARY DNA-directed RNA Polymerase II (Pol II) is one of the most important molecules in biology. Pol II is highly- conserved among eukaryotic organisms and plays a fundamental role in cellular life; specifically, the transcription of genes into messenger RNA. Structural studies of Pol II have been very successful and have allowed snapshots of Pol II in its apo form, in the process of initiation and elongation, during backtracking or paused by DNA lesions. Moreover, single particle cryo-electron microscopy structures of Pol II in complex with the general transcription factors (preinitiation complex), and with elongation factors have provided views of the complexities of the initiation and elongation steps of transcription. A molecular picture of the role that individual factors play during transcription initiation and elongation is beginning to emerge and has generated tremendous progress towards our understanding of gene processing and regulation. Conversely, the molecular details of nucleotide addition and the roles of conformational changes by conserved and essential domains such as the so called “trigger loop” or “bridge helix” in substrate selection and catalysis are not fully understood. Furthermore, pioneering studies are at lower resolution and miss critical information to inform a complete enzymatic mechanism. This project, through extensive technology development and innovation in structural approaches to Pol II, describes the foundation to reveal the active site rearrangements within Pol II leading to catalysis and subsequent translocation. The studies proposed employ a combination of state of the art technologies including time resolved X-ray crystallography, free electron laser and single particle cryo-electron microscopy experiments to elucidate the time evolution of the molecular events during Pol II transcriptional elongation. Importantly, orthogonal approaches are developed to validate results from multiple independent methodologies. The Pol II system represents a model for all cellular RNA polymerases and results obtained will represent foundational models with which Pol I, Pol III, and prokaryotic RNA polymerases may be compared. Insight into the Pol II catalytic mechanism and determination of how activity-altering mutants alter it will provide the framework for understanding ho regulatory factors that target the Pol II active site might work.

项目概要 DNA 引导的 RNA 聚合酶 II (Pol II) 是生物学中最重要的分子之一。在真核生物中保守并在细胞生命中发挥重要作用； Pol II 的基因转录为信使 RNA 的结构研究非常成功并已取得进展。快照允许 Pol II 以其 apo 形式、在起始和延伸过程中、在回溯或此外，复合体中 Pol II 的单粒子冷冻电子显微镜结构。与一般转录因子（起始复合物）和延伸因子一起提供转录起始和延伸步骤复杂性的视图。各个因素在转录起始和延伸过程中所起的作用开始出现并已在我们对基因加工和调控的理解方面取得了巨大进展。核苷酸添加的分子细节以及保守和构象变化的作用底物选择和催化中的重要领域，例如所谓的“触发环”或“桥螺旋” 此外，开创性研究的分辨率较低且错过关键。该项目通过广泛的技术提供了完整的酶促机制信息。 Pol II 结构方法的发展和创新，描述了揭示活性的基础研究提出，Pol II 内的位点重排会导致催化和随后的易位。采用最先进的技术组合，包括时间分辨 X 射线晶体学、免费电子激光和单粒子冷冻电子显微镜实验阐明了重要的是，开发了正交方法。验证多种独立方法的结果 Pol II 系统代表了所有方法的模型。细胞 RNA 聚合酶和获得的结果将代表 Pol I、Pol III、和原核 RNA 聚合酶可以深入了解 Pol II 催化机制。确定改变活性的突变体如何改变它将为理解何为提供框架针对 Pol II 活性位点的监管因素可能会起作用。