Structural Studies of RNA Polymerase II Transcription Initiation and Elongation

项目总结 DNA导向的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，在细胞生命中起着基本作用；具体地说， 基因转录成信使核糖核酸。POL II的结构研究非常成功，并已 在启动和延长过程中、在回溯期间或 因DNA损伤而暂停。此外，Pol II在络合物中的单颗粒冷冻电子显微镜结构 与一般转录因子(预起始复合体)、以及与延伸因子已提供 对转录起始和延伸步骤的复杂性的看法。这是一张分子图像 个别因子在转录起始和延伸过程中发挥的作用开始显现，并已 在我们对基因处理和调控的理解方面产生了巨大的进步。相反， 核苷酸加成的分子细节和保守的构象变化的作用 底物选择和催化中的基本结构域，如所谓的“触发环”或“桥螺旋” 还没有完全被理解。此外，开创性研究的分辨率较低，错过了关键 信息，以告知一个完整的酶机制。这个项目，通过广泛的技术 在POL结构方法的发展和创新中，描述了积极揭示的基础 POL II内的位点重排导致催化和随后的易位。这项研究建议 采用包括时间分辨X射线结晶学在内的最先进技术的组合，免费 用电子激光和单粒子冷冻电子显微镜实验阐明 POL II转录延伸过程中的分子事件。重要的是，发展了正交化方法。 以验证来自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， 与原核生物的RNA聚合酶进行比较。深入了解POL II的催化机理和 确定改变活性的突变体如何改变它将为理解ho提供框架。 针对POL II活动站点的监管因素可能会起作用。

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.