"Mechanisms of RNA Polymerase II initiation"

项目总结 真核生物的转录是在数十到数百种蛋白质的协调作用之后开始的。 这些活动导致RNA聚合酶II(POL II)被招募到核心启动子和随后的 启动过程。在起始过程中，Pol II必须被定向到转录起始点(TSS)，然后是TSS 识别、初始磷酸二酯键的形成和启动子清除。POL II的作用机制 启动与TSS的接合以及这一过程如何确定总体启动子产量出人意料地差 明白了。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 使用了TS。有了一个协作小组，我们希望确定Pol II 参与启动子的启动以及启动子结构对启动的影响。在萌芽酵母中，Pol II 扫描tss，这使我们能够合理化启动和启动子中的生化活动 架构可能决定启动级别。我们已经通过一种我们称之为枪击的模型来使引爆合理化 将启动概率与POL II活性中心和Pol II活动之间的合作联系起来的图库 TFIIH，驱动扫描的假定马达。我们设计了遗传、基因组和生化 探索和测试射击馆模型的实验。我们的方法包括定量建模 在所有酵母启动子上启动，以确定它们如何可能是唯一的或什么启动子特征控制 表达级别。我们将建立裂殖酵母和黑腹果蝇作为模型 直接测试启动子扫描的保守性作为POL II启动的机制。我们的实验将 揭示真核生物中是否存在普遍的启动机制。我们预计将大幅增加 我们根据POL II和TFIIH的生化特性预测启动子产量的能力，我们对 启动子结构，以及它们与潜在序列的相互作用，每一个都是 基因在所有真核生物中的表达。

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.