Mechanisms of RNA Polymerase II transcription

项目摘要 真核生物中基因的转录涉及几十种蛋白质的协调，如果不是几百种的话， RNA聚合酶II（Pol II）转录的每一步：起始、延伸和终止。这些步骤发生在 动态地在包装成染色质的DNA模板上，并且与宿主的共转录的 事件拟议的研究广泛涉及两个主要主题- Pol II起始和Pol II延伸。第一 转录的起始步骤涉及选择转录起始位点（TSS）。跨真核生物的启动子 可以有多个TSS，这些TSS可以在广泛的级别上使用。该项目将确定 全面研究了模式生物酿酒酵母中TSS选择的决定因素， 建立在其他真核生物中起始对改变的Pol II催化活性敏感的程度， 对真核生物不同起始机制的重要检验。这些研究利用大规模并行 启动子测定和TSS使用的全基因组分析。启动发生后，共转录事件运行 从RNA加工（包括加帽、剪接和RNA切割）到修饰或 模板染色质的重塑。这个项目将揭示如何延长Pol II工程在一个基本的 Pol II和共转录剪接之间的水平和探针连接;何时发生以及如何发生 由Pol II延伸率控制。拟议的工作是独特的定位在广泛的 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 physiology.第一个S。所采用的酿酒酵母模型具有几个关键的优势，以利用这些 目的-在其他系统中难以匹配的规模上的遗传学， 生化分析基因表达机制将在许多尺度上进行探索，从确定基因的作用， Pol II活性位点中对于适当的催化控制至关重要的单个氨基酸， 由不同的遗传或化学扰动引起的转录组。在Pol II研究中独一无二 机制，Pol II结构分析与Pol II活性位点的深度突变扫描的耦合 将为这些酶的功能和进化提供新的见解。Pol II的保护及其 调节机制支持获得的结果与每个真核细胞（包括人类）的相关性 我们现在认识到一种复杂的综合征与Pol II大亚基本身的突变有关。

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.