Mechanisms of RNA Polymerase II transcription

项目总结 真核生物中基因的转录涉及到数十个(如果不是数百个)蛋白质的编排，使 RNA聚合酶II(POL II)转录的每一步：起始、延长和终止。这些步骤会发生 动态地将DNA模板打包成染色质，并巧合地与许多共转录 事件。拟议的研究大致涉及两个主题--Pol II的启动和Pol II的延伸。第一 转录的步骤，即起始，涉及选择转录起始位点(TSS)。真核生物中的启动子 可以有多个TS，并且这些TS可以在广泛的级别上使用。该项目将确定 模式生物酿酒酵母和酿酒酵母TSS选择的决定因素 确定在其他真核生物中，启动对改变的POL II催化活性的敏感程度，以及 对真核生物启动的不同机制的重要测试。这样的研究利用了大量的平行 启动子分析和TSS使用的全基因组分析。在启动发生后，共转录事件运行 从RNA加工包括封端、剪接和RNA切割，到修饰或 模板染色质重塑。这个项目将揭示Pol II的延伸率是如何在一个基本的 POL II和共转录剪接之间的水平和探针连接；它发生的时间和方式 由POL II延伸率控制。建议的工作在广泛的范围内具有独特的定位 生物学，从POL II功能的基本机制到POL II的变化如何导致细胞变化 生理学。所采用的原始酿酒酵母模型有几个关键的优势可供利用 目的-遗传学的规模在其他系统中难以匹敌，并可适应结构和 生化分析。基因表达机制将在多个尺度上进行探索，从确定 Pol II活性部位中对适当的催化控制至关重要的个别氨基酸，对 转录体由不同的遗传或化学干扰引起的转录体在POL II研究中独树一帜 机制、POL II结构分析与POL II活性部位深度突变扫描的结合 将为这些酶的功能和进化提供新的见解。POL II及其守恒 监管机制支持所获得的结果与包括人类在内的每个真核细胞的相关性。 我们现在认识到一种复杂的综合症，与POL II自身大亚基的突变有关。

PROJECT SUMMARY Transcription of genes in eukaryotes involves the orchestration of dozens if not hundreds of proteins enabling each step in RNA Polymerase II (Pol II) transcription: initiation, elongation, and termination. These steps occur dynamically on DNA templates packaged into chromatin and coincidentally with a host of cotranscriptional events. The proposed studies broadly involve two main themes – Pol II initiation and Pol II elongation. The first step in transcription, initiation, involves selecting a transcription start site (TSS). Promoters across eukaryotes can have multiple TSSs and these can be used at a wide range of levels. This project will identify comprehensively the determinants of TSS selection in the model organism Saccharomyces cerevisiae and establish the extent to which initiation is sensitive to altered Pol II catalytic activity in other eukaryotes, an important test of distinct mechanisms for initiation in eukaryotes. Such studies utilize massively parallel promoter assays and genome-wide analysis of TSS usage. After initiation occurs, cotranscriptional events run the gamut from RNA processing encompassing capping, splicing, and RNA cleavage, to modification or remodeling of template chromatin. This project will uncover how elongation by Pol II works at a fundamental level and probe connections between Pol II and cotranscriptional splicing; when it occurs and how it is controlled by Pol II elongation rate. The work proposed is uniquely positioned across a wide spectrum of biology, from fundamental mechanisms of Pol II function, to how changes to Pol II result in changes to cellular physiology. The primary S. cerevisiae model employed has several critical advantages to exploit for these purposes – genetics on a scale difficult to match in other systems and amenability to structural and biochemical analyses. Gene expression mechanisms will be probed at many scales, from determining roles of individual amino acids in the Pol II active site that are essential for proper catalytic control, to the changes in transcriptomes caused by distinct genetic or chemical perturbations. Unique among studies on Pol II mechanisms, the coupling of Pol II structural analyses with deep mutational scanning of the Pol II active site will provide novel insights for how these enzymes function and evolve. The conservation of Pol II and its regulatory machinery support the pertinence of results obtained to every eukaryotic cell, including humans where we now recognize a complex syndrome linked to mutations in the large subunit of Pol II itself.