RNA POLYMERASE I TRANSCRIPTION & REGULATION MECHANISMS

描述(来自申请表)：这个实验室是一个主要的实验室 对我们理解rRNA转录机制的贡献 倡导和规范。然而，我们研究的实验系统 没有表现出更高的真核rRNA转录的一些特征， 特别是，它不需要上游激活剂或上游启动子 元素。因为酿酒酵母不需要这些，而且更有非常 简单而强大的遗传学，酵母的生化和遗传学研究是 建议。在过去的十年中，rrna转录的遗传学 在酵母中有效利用，通过分离定义三个基因 转录因子UAF、Cf和Rrn3p，这是除了 聚合酶I和激活转录的TATA结合因子。这个 该系统的生物化学明显落后于遗传学， 留下了一些机械性的问题没有得到回答。这些将是 调查过了。已确定的和预引发的络合物的结构将 通过一系列方法来揭示，包括五种足迹方法和 位点特异性DNA-蛋白质光交联，药物抑制研究 TBP突变体的拓扑分析和遗传筛选。这些研究将 确定预引发络合物的结构，确定沟槽 主要由蛋白质接触的DNA，以及DNA在这些 复合体。酵母双杂交分析将揭示蛋白质-蛋白质 络合物中的相互作用。TBP在活化过程中的机制作用 转录将被阐明，重点是它的相互作用机制。 以及由此产生的DNA拓扑结构的变化。此外，功能 将对其中几个因素的作用机制进行研究。尤其是， 转录因子参与rRNA调控的机制 转录，Rrn3p，将被确定；检测以确定它是否参与 将使用聚合酶招募、DNA融化或启动子清除。核糖核酸 聚合酶I及其相互作用的转录因子亚基 其他调解Pol I招募的方法将通过生化和 从基因上来说。聚合酶亚基或Rrn3p修饰在血管内皮细胞癌中的潜在作用 监管将受到调查。这些研究将产生最完整的 描述任何真核rRNA的生物化学和遗传学 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.