Subunit interactions of components of yeast Paf1 complex

描述（由申请人提供）：RNA聚合酶II （pol II）对mRNA合成的调控对生物体的正常发育、稳态和功能至关重要；失调会导致包括癌症在内的严重疾病。Pol II是一个支架，其他对mrna转录和转录后加工很重要的因子，包括一般转录因子、tata结合蛋白及其相关因子、中介复合物、capping酶复合物和裂解/聚腺苷化因子，都聚集在这个支架上。除了这些因子外，与启动子和基因编码区域的pol II相关的Paf1复合物（Paf1C）正在成为协调转录和转录后因子结合和活性的重要参与者。酵母Paf1C含有Paf1， Ctr9, Rtf1， Leo1和Cdc73，现在在遗传和物理上与起始和延伸因子以及mRNA的转录后加工联系在一起。酵母Paf1因子的突变不是致命的，但会导致严重的多效表型，并导致聚腺苷化和mrna子集丰度的变化。人类Cdc73同源物（parafibromin）的突变导致人类甲状旁腺和颌骨肿瘤，将Paf1C定义为人类肿瘤抑制因子。我们仍然不知道Paf1C组分的相互联系，也不知道该复合物如何与pol II相关联。我们也没有与其他转录和RNA加工因子直接的蛋白质/蛋白质接触的定义。酵母蛋白-蛋白相互作用的两个基因组规模分析没有发现任何这些相互作用，部分原因是未能在筛选中包括Paf1C因子的正确编码区域。该项目提出了Paf1C成员之间、彼此之间、与pol II亚基之间以及与其他功能相关因子之间的蛋白-蛋白相互作用的定向测试。酵母双杂交技术将用于检测蛋白质在体内的相互作用。一种互补的体外方法将使用带有标记蛋白对的酵母菌株，在野生型和Paf1C因子突变型酵母菌株中寻找蛋白质的共免疫沉淀。我们的研究结果将建立酵母Paf1C组织及其相互作用的模型，这对于理解这种重要的复合物如何在人类中作为肿瘤抑制因子发挥作用至关重要。模型系统的简单性和所使用的技术保证了本科生将能够对本提案中提出的重要问题做出重大贡献。

DESCRIPTION (provided by applicant): Regulation of mRNA synthesis by RNA polymerase II (pol II) is critical for proper development, homeostasis and functioning of organisms; misregulation contributes to serious diseases including cancer. Pol II is a scaffold upon which other factors important for transcription and post-transcriptional processing of mRNAs assemble, including the general transcription factors, TATA-binding protein and its associated factors, the Mediator complex, the capping enzyme complex and the cleavage/polyadenylation factors. In addition to these factors, the Paf1 complex (Paf1C), associated with pol II at the promoter and in coding regions of genes, is emerging as an important player orchestrating the binding and activity of transcription and post- transcriptional factors. Containing Paf1, Ctr9, Rtf1, Leo1 and Cdc73, the yeast Paf1C is now linked genetically and physically to initiation and elongation factors and to post-transcriptional processing of mRNA. Mutation of yeast Paf1 factors Is not lethal, but causes severe, pleiotropic phenotypes, and results in changes in polyadenylation and the abundance of a subset of mRNAs. Mutation of the human Cdc73 homolog (parafibromin) results in human parathyroid and jaw tumors defining the Paf1C as a human tumor suppressor. We still do not know the interconnections of the Paf1C components or how the complex associates with pol II. Nor do we have a definition of the direct protein/protein contacts made with other transcription and RNA processing factors. Two genome scale analyses of yeast protein-protein interactions have not identified any of these interactions, due in part to failure to include the correct coding regions for the Paf1C factors in the screens. This project proposes a directed test of protein-protein interactions of the members of the Paf1C, with each other, with pol II subunits, and with other functionally linked factors. The yeast two-hybrid technique will be used to detect interactions of proteins in vivo. A complementary in vitro approach will use yeast strains with pairs of tagged proteins to look for co-immunoprecipitation of proteins in both wild-type and Paf1C factor mutant yeast strains. Our results will build a model of the organization of the yeast Paf1C and its interactions critical for understanding how this important complex functions as a tumor suppressor in humans. The simplicity of the model system and techniques to be used assure that under- graduates will be able to make significant contributions to the important questions asked in this proposal.