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：
10385793
负责人：
Guillermo Alberto Calero
金额：
$ 27.34万
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/10385793
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 II 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 Positioning Attribute Process Publishing RNA 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 innovation insight millisecond movie mutant particle scaffold 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.

项目摘要 DNA指导的RNA聚合酶II（POL II）是生物学中最重要的分子之一。 Pol II高度 - 在真核生物中配置并在细胞生活中起着基本作用；具体来说，将基因转录到信使RNA中。 Pol II的结构研究非常成功，并且在启动和伸长过程中，在回溯或被DNA病变暂停。此外，POL II的单个粒子冷冻电子显微镜结构在复合物中随着一般的转录因子（预启用复合物）以及伸长因子的提供对倡议的复杂性和转录步骤的复杂性的看法。一幅分子图片单个因素在转录启动和伸长过程中所扮演的角色开始出现，并且具有在我们对基因加工和调节的理解方面产生了巨大的进步。反过来，核苷酸添加的分子细节以及保守和构象变化的作用基板选择和催化中所谓的“触发循环”或“桥螺旋”之类的必需域不完全理解。此外，开创性的研究处于较低的分辨率，错过了关键信息以告知完整的酶促机制。这个项目，通过广泛的技术 Pol II结构方法的发展和创新，描述了揭示活跃的基础 POL II内的现场重排导致催化和随后的易位。研究提出员工一种艺术技术的结合，包括时间解决的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，以阐明 POL II转录伸长期间的分子事件。重要的是，开发了正交方法从多种独立方法验证结果。 Pol II系统代表了所有的模型细胞RNA聚合酶和获得的结果将代表使用Pol I，Pol III，可以比较核酸核RNA聚合酶。深入了解pol II催化机制和确定改变活动突变体如何改变它将为理解HO提供框架针对POL II主动部位的监管因素可能会起作用。