RNA POLYMERASE II TRANSCRIPTION INITIATION COMPLEX

RNA 聚合酶 II 转录起始复合物

• 批准号: 2734715
• 负责人: Stephen Buratowski
• 金额: $ 26.96万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734715
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Saccharomyces cerevisiae; enzyme activity; fungal genetics; gel mobility shift assay; gene interaction; genetic promoter element; genetic transcription; immunoprecipitation; intermolecular interaction; mutant; protein structure function; site directed mutagenesis; suppressor mutations; transcription factor

The goal of this proposal is to explore the structure 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 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. Currently, mutants have been isolated in the genes encoding the TATA Binding Protein (TBP), TFIIB, the Tfb1 subunit of TFIIH, the small subunit of TFIIF, and the large subunit of TFIIE. This analysis will be extended to the remaining subunits of TFIID (TAFs), TFIIE, TFIIF, and TFIIH. Many of the basal transcription factors interact with each other, so biochemical assays such as in vitro transcription, native gel electrophoresis, 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.

这项计划的目标是探索RNA的结构 聚合酶II转录起始复合物，其使用以下的组合 遗传和生化分析。 酵母酿酒酵母 提供了一种理想的在体内和体内进行协同的系统， 在其他模型系统中不可行的体外研究。 酵母 转录机制与哺乳动物细胞中的转录机制高度同源， 所以一个系统中的发现可以扩展到另一个系统。 研究 这里提出的将有助于我们理解基因表达， 这个过程中的缺陷如何导致癌症或发育 异常 此外，了解具体细节， 酵母中的转录可以为设计 抗真菌药物 特异性基础转录的基因将被诱变和引入 转化成酵母细胞 赋予有趣表型的突变基因（例如， 高或低温度敏感性，不能在某些培养基上生长） 将得到进一步表征。 目前，已经分离出突变体， 编码TATA结合蛋白（TBP）、TFIIB、Tfb 1 TFIIH的亚基、TFIIF的小亚基和TFIIF的大亚基。 TFIIE。 这一分析将扩展到TFIID的其余子单元 （TAF）、TFIIE、TFIIF和TFIIH。 许多基础转录因子相互作用， 生物化学测定，例如体外转录、天然凝胶电泳（native gel 电泳和免疫共沉淀将用于测定 蛋白质与突变蛋白质的相互作用。 缺陷 特定的生化功能将与基因突变相关， 转录因子的特定残基或区域。 是 预计这种方法将允许特定的活动和蛋白质- 蛋白质接触进行研究。 在一个转录因子突变破坏相互作用的情况下 与第二因子结合从而导致条件表型， 第二因子基因中的抑制子将被分离， 在限制温度下生长。 同样， 将通过检测蛋白质-蛋白质， 体外相互作用 此外，随机诱变将用于分离 突变转录因子的第二位点抑制子。 的蛋白质 被鉴定为抑制因子（无论是已知的因子还是新的蛋白质）， 它们与转录的相互作用 机械.