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合成的调节对于生物体的正常发育、稳态和功能至关重要;调节不当会导致严重疾病，包括癌症。Pol II是一种支架，在其上组装对mRNA的转录和转录后加工重要的其他因子，包括一般转录因子、TATA结合蛋白及其相关因子、介体复合物、加帽酶复合物和切割/聚腺苷酸化因子。除了这些因子之外，在基因的启动子和编码区与pol II相关的Paf 1复合物（Paf 1C）正在成为协调转录和转录后因子的结合和活性的重要参与者。含有Paf 1，Ctr 9，Rtf 1，Leo 1和Cdc 73，酵母Paf 1C现在在遗传和物理上与起始和延伸因子以及mRNA的转录后加工相关联。酵母Paf 1因子的突变不是致命的，但会导致严重的多效性表型，并导致多聚腺苷酸化和mRNA子集丰度的变化。人类Cdc 73同源物（parafibromin）的突变导致人类甲状旁腺和颌骨肿瘤，将Paf 1C定义为人类肿瘤抑制因子。我们仍然不知道Paf 1C组分的相互联系，也不知道复合物如何与pol II结合。我们也没有一个直接的蛋白质/蛋白质接触与其他转录和RNA加工因子的定义。两个基因组规模的酵母蛋白质-蛋白质相互作用的分析没有确定任何这些相互作用，部分原因是未能包括正确的编码区的Paf 1C因子在屏幕上。该项目提出了一个直接测试的蛋白质-蛋白质相互作用的成员的Paf 1C，彼此之间，与pol II亚基，并与其他功能相关的因素。酵母双杂交技术将用于检测体内蛋白质的相互作用。一种互补的体外方法将使用带有成对标记蛋白的酵母菌株来寻找野生型和Paf 1C因子突变酵母菌株中蛋白质的免疫共沉淀。我们的研究结果将建立一个酵母Paf 1C组织及其相互作用的模型，这对于理解这个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.