RNA POLYMERASE II TRANSCRIPTION INITIATION COMPLEX

RNA 聚合酶 II 转录起始复合物

• 批准号: 6519464
• 负责人: Stephen Buratowski
• 金额: $ 29.24万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-06 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519464
• 关键词: DNA binding protein; DNA directed RNA polymerase; Saccharomyces cerevisiae; enzyme activity; fungal genetics; gene interaction; genetic promoter element; genetic transcription; protein protein interaction; protein structure function; site directed mutagenesis; suppressor mutations; transcription factor

The goal of this proposal is to explore the structure and function of the RNA polymerase II transcription initiation complex using a combination of genetic and biochemical assays. The yeast Saccharomyces cerevisiae provides an ideal system for carrying out coordinated in vivo and in vitro studies that are not feasible in other model systems. The yeast transcription machinery is highly homologous to that in mammalian cells, so findings in one system can be extended to the other. The studies proposed here will contribute to our understanding of gene expression and of how defects in this process lead to cancer or developmental abnormalities. In addition, understanding the specific details of transcription in yeast may provide useful information for designing antifungal drugs. Genes for specific basal transcription factors will be mutagenized and introduced into yeast cells. Mutant genes that confer interesting phenotypes (e.g. high or low temperature sensitivity, inability to grow on certain media) will be further characterized. Mutants will be analyzed in the genes encoding the TATA Binding Protein (TBP), the TAF subunits of TFIID, TFIIB, the Kin28 subunit of TFIIH, and both subunits of TFIIE. In addition, two new bromodomain-containing proteins that interact with TFIID have been isolated and will be characterized for their role in transcription. Many of the basal transcription factors interact with each other, so biochemical assays such as in vitro transcription, native gel electrophoresis, immobilized template assay, and co- immunoprecipitation will be used to assay protein-protein interactions with the mutant proteins. Defects in specific biochemical functions will be correlated with mutations in specific residues or regions of the transcription factors. It is expected that this approach will allow specific activities and protein-protein contacts to be studied. In cases where mutations in one transcription factor disrupt interactions with a second factor and thereby cause a conditional phenotype, suppressors in the second factor gene will be isolated that restore growth at the restrictive temperature. Again, the in vivo behavior of the mutant-suppressor pairs will be explored by testing protein-protein interactions in vitro. Also, random mutagenesis will be used to isolate second-site suppressors of mutant transcription factors. The proteins identified as suppressors (whether known factors or new proteins) will be characterized as to their interactions with the transcription machinery.

该建议的目标是利用遗传和生化分析相结合的方法来探索RNA聚合酶II转录起始复合体的结构和功能。酿酒酵母为进行体内和体外协同研究提供了理想的系统，这在其他模型系统中是不可行的。酵母转录机制与哺乳动物细胞的转录机制高度同源，因此在一个系统中的发现可以延伸到另一个系统。这里提出的研究将有助于我们理解基因表达以及这一过程中的缺陷如何导致癌症或发育异常。此外，了解酵母中转录的具体细节可能会为设计抗真菌药物提供有用的信息。特定基础转录因子的基因将被诱变并导入酵母细胞。赋予有趣表型的突变基因(例如，高或低温度敏感性，不能在某些介质上生长)将被进一步表征。突变将在编码TATA结合蛋白(TBP)、TFIID的TAF亚基、TFIIB、TFIIH的Kin28亚基以及TFIIE的两个亚基的基因中进行分析。此外，还分离出了两个新的与TFIID相互作用的含溴结构域的蛋白质，并将对其在转录中的作用进行表征。许多基础转录因子相互作用，因此将使用体外转录、天然凝胶电泳法、固定化模板实验和免疫共沉淀等生物化学方法来检测蛋白质与突变蛋白质之间的相互作用。特定生化功能的缺陷将与转录因子的特定残基或区域的突变相关。预计这种方法将允许研究特定的活动和蛋白质-蛋白质接触。在一个转录因子的突变破坏与第二个因子的相互作用从而导致条件表型的情况下，第二个因子基因中的抑制子将被分离出来，从而在限制性温度下恢复生长。再次，我们将通过在体外测试蛋白质-蛋白质的相互作用来探索突变体-抑制子对的体内行为。此外，随机突变将被用来分离突变转录因子的第二位点抑制子。被确认为抑制子的蛋白质(无论是已知的因子还是新的蛋白质)将根据它们与转录机制的相互作用进行表征。