RNA POLYMERASE I TRANSCRIPTION & REGULATION MECHANISMS

描述（来自应用程序）：该实验室一直是一个主要的 有助于我们理解rRNA转录的机制 启动和调节。然而，我们所研究的实验系统 不表现出高等真核生物rRNA转录的某些特征， 特别地，它不需要上游激活剂或上游启动子 元素因为S.酿酒厂不需要这些，并且还具有非常大的 简单而强大的遗传学，酵母的生物化学和遗传学研究， 提出了在过去的十年里，rRNA转录的遗传学已经被 在酵母中有效利用，分离出定义三个 转录因子，UAF，CF和Rrn 3 p，这是需要除了 聚合酶I和TATA结合因子用于激活转录。的 系统的生物化学已经明显落后于遗传学， 留下了许多机械性的问题没有答案。这些将是 研究了定向复合物和前起始复合物的结构将 可以通过一系列方法来揭示，包括五种足迹方法， 位点特异性DNA-蛋白质光交联，药物抑制研究，DNA 拓扑分析和TBP突变体的遗传筛选。这些研究将 确定前起始复合体的结构，确定 主要与蛋白质接触的DNA，以及这些DNA在蛋白质中的路径 配合物酵母双杂交分析将揭示 复合体中的相互作用。TBP在活化细胞中的机制作用 转录将阐明，重点是其相互作用的机制 与DNA结合，导致DNA拓扑结构的改变。此外，功能 将研究这些因素中的几个的机制。特别是 转录因子参与rRNA调控的机制 转录，Rrn 3 p，将被确定;测定，以确定它是否介导 将使用聚合酶募集、DNA解链或启动子清除。的RNA 聚合酶I和转录因子亚单位，与每个相互作用 其他介导pol I募集的蛋白质将通过生物化学和 遗传的聚合酶亚基或Rrn 3 p修饰在细胞凋亡中的潜在作用 监管将被调查。这些研究将产生最完整的 任何真核生物rRNA的生物化学和遗传学描述 转录系统

DESCRIPTION (from the application): This laboratory has been a major contributor to our understanding of the mechanism of rRNA transcription initiation and regulation. However, the experimental system we have studied does not exhibit some characteristics of higher eukaryotic rRNA transcription, in particular, it does not require an upstream activator or upstream promoter elements. Because S. cerevisiae does not require these, and further has very facile and powerful genetics, a biochemical and genetic study of yeast is proposed. Over the past decade, the genetics of rRNA transcription has been effectively exploited in yeast, with isolation of genes defining three transcription factors, UAF, CF, and Rrn3p, which are needed in addition to polymerase I and TATA-binding factor for activated transcription. The biochemistry of the system has lagged significantly behind the genetics, leaving a number of mechanistic questions unanswered. These will be investigated. The structure of the committed and preinitiation complexes will be revealed by a battery of approaches, including five footprinting methods and site-specific DNA-protein photo-cross-linking, drug inhibition studies, DNA topological analyses and genetic screens for TBP mutants. These studies will determine the architecture of the preinitiation complexes, determine the groove of the DNA primarily contacted by proteins, and the path of the DNA in these complexes. Yeast two-hybrid analyses will reveal the protein-protein interactions in the complexes. The mechanistic role of TBP in activated transcription will be elucidated, with emphasis on its mechanism of interaction with DNA, and resulting DNA topology changes. In addition, the functional mechanism of several of these factors will be investigated. In particular, the mechanism of the transcription factor implicated in the regulation of rRNA transcription, Rrn3p, will be determined; assays to determine if it mediates polymerase recruitment, DNA melting or promoter clearance will be used. The RNA polymerase I and the transcription factor subunits that interact with each other to mediate pol I recruitment will be determined both biochemically and genetically. The potential role of polymerase subunit or Rrn3p modification in regulation will be investigated. These studies will yield the most complete description of the biochemistry and genetics of any eukaryotic rRNA transcription system.