"Mechanisms of RNA Polymerase II initiation"

项目摘要 真核生物中的转录是在几十到几百个蛋白质的协调作用后启动的。 这些活性导致RNA聚合酶II（Pol II）募集到核心启动子，并且随后的启动子被激活。 启动过程。在起始过程中，Pol II必须被引导至转录起始位点（TSS），随后是TSS 识别、初始磷酸二酯键形成和启动子清除。Pol II的机制 启动涉及TSS，并且该过程如何决定总体启动子输出令人惊讶地差 明白真核生物中的大多数启动子以广泛或分散的方式启动转录，其中多个启动子在转录过程中起重要作用。 使用TSS。随着一个合作小组的到位，我们希望确定Pol II的机制， 启动子结构如何影响启动。在芽殖酵母中，Pol II 扫描TSS，这使我们能够合理化启动子和启动子中的生化活性， 架构可以确定发起级别。我们通过一个我们称之为“射击”的模型， 将起始概率与Pol II活性位点和 TFIIH，驱动扫描的假定马达。我们设计了遗传学基因组学和生物化学 实验来探索和测试射击馆模型。我们的方法包括定量建模， 启动所有酵母启动子，以确定它们如何可能是独特的或什么启动子特征控制 表达水平。我们将建立粟酒裂殖酵母和黑腹果蝇作为模型， 启动子扫描作为Pol II起始机制的保守性的直接测试。我们的实验将 揭示真核生物中是否存在普遍的起始机制。我们预计将大大增加 我们预测启动子输出的能力基于Pol II和TFIIH的生化特性，我们对 启动子结构，以及它们与潜在序列的相互作用，其中每一个都是 所有真核生物的基因表达。

PROJECT SUMMARY Transcription in eukaryotes is initiated following the coordinated action of dozens to hundreds of proteins. These activities result in the recruitment of RNA Polymerase II (Pol II) to core promoters and the subsequent initiation process. During initiation, Pol II must be directed to the transcription start site (TSS) followed by TSS recognition, initial phosphodiester bond formation, and promoter clearance. The mechanism by which Pol II initiation engages the TSS and how this process determines overall promoter output is surprisingly poorly understood. Most promoters in eukaryotes initiate transcription in a broad or dispersed fashion where multiple TSSs are used. With a collaborative team in place, we wish to determine the mechanism by which Pol II engages TSSs in initiation and how initiation is affected by promoter architecture. In budding yeast, Pol II scans for TSSs and this allows us to rationalize how biochemical activities in initiation and promoter architecture may determine initiation levels. We have rationalized initiation by a model we call the Shooting Gallery that relates initiation probability to the cooperation between the Pol II active site and the activity of TFIIH, the putative motor that drives scanning. We have designed genetic, genomic, and biochemical experiments to explore and test the Shooting Gallery model. Our approaches include quantitative modeling of initiation at all yeast promoters to determine how they might be unique or what promoter features control expression levels. We will establish Schizosaccharomyces pombe and Drosophila melanogaster as models for direct tests of the conservation of promoter scanning as a mechanism for Pol II initiation. Our experiments will reveal whether there are universal initiation mechanisms in eukaryotes or not. We expect to greatly increase our power to predict promoter output based on biochemical properties of Pol II and TFIIH, our understanding of promoter architecture, and their interplay with underlying sequence, each of which are fundamental features of gene expression in all eukaryotes.