Structural Studies of RNA Polymerase II Transcription Initiation and Elongation

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是高度- 在真核生物中保守，在细胞生命中起着重要作用;具体来说， 基因转录成信使RNA。Pol II的结构研究非常成功， 允许Pol II在其apo形式中的快照，在起始和延伸过程中，在回溯或 因DNA损伤而暂停此外，在复合物中的Pol II的单颗粒冷冻电子显微镜结构， 与一般转录因子（前起始复合物），并与延伸因子提供了 对转录起始和延伸步骤的复杂性的看法。一张分子图像 单个因子在转录起始和延伸过程中所起的作用开始显现， 对我们理解基因加工和调控产生了巨大的进步。相反地， 核苷酸添加的分子细节和构象变化的作用， 在底物选择和催化中的基本结构域，例如所谓的“触发环”或“桥螺旋 还没有被完全理解。此外，开创性研究的分辨率较低，且未达到临界值 提供完整的酶机制信息。这个项目通过广泛的技术 发展和创新的结构方法，以波尔II，描述了基础，以揭示积极的 Pol II内的位点重排导致催化和随后的易位。建议的研究 采用最先进技术的组合，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，以阐明时间演变的 Pol II转录延伸过程中的分子事件。重要的是，开发了正交方法 验证多个独立方法的结果。Pol II系统代表了所有人的模式。 细胞RNA聚合酶和获得的结果将代表Pol I，Pol III， 和原核RNA聚合酶可以进行比较。深入了解Pol II催化机制， 确定活性改变突变体是如何改变它的，这将为理解基因的功能提供一个框架。 靶向Pol II活性位点的调节因子可能起作用。

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.