BIOCHEMISTRY OF SPECIFIC INITIATION BY RNA POLYMERASE II

RNA 聚合酶 II 特异性引发的生物化学

• 批准号: 2415223
• 负责人: MICHAEL H. SAYRE
• 金额: $ 12.17万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415223
• 关键词: DNA directed RNA polymerase; DNA footprinting; RNA biosynthesis; adenosinetriphosphatase; enzyme complex; enzyme mechanism; enzyme reconstitution; fungal genetics; genetic promoter element; genetic transcription; helicase; messenger RNA; nuclear runoff assay; phosphoprotein phosphatase; phosphorylation; protein kinase; protein purification; protein sequence; recombinant DNA; site directed mutagenesis; temperature sensitive mutant; transcription factor

The ultimate goal of this research is to understand how eukaryotic messenger RNA synthesis is regulated at the molecular level. An important first step will be to determine the enzymatic mechanism of initiation catalyzed by RNA polymerase II and a set of "initiation factors" (termed a, b, d, e and g) purified from yeast. Those proteins are now available in quantities sufficient for mechanistic studies. Factor b fails to support transcription reconstituted with pure forms of polymerase and the other initiation factors, but a factor b "holoenzyme" with that capacity has been isolated. Likewise, a "native" polymerase- factor g complex with specific activity much higher than that of polymerase or factor g purified separately has been identified. These holoenzymes will be purified to establish a fully defined system capable of efficient template utilization. The initiation mechanism will be investigated through physical and enzymological studies. The structure and dynamics of protein-DNA assemblies will be determined by nondenaturing gel electrophoresis and chemical or enzymatic "footprinting" methods. Single- and multiple-round transcription protocols will be used to test whether phosphorylation and dephosphorylation of RNA polymerase II are major determinants of reaction efficiency. Factor a, the yeast homologue of the human initiation factor TFIIE, will be mutagenized and purified from E. coli to determine if conserved domains in its primary structure are important for transcription activity. Temperature-sensitive mutants will be isolated and analyzed to determine if factor a/TFIIE is required for RNA synthesis in vivo.

这项研究的最终目标是了解真核生物如何 信使RNA的合成在分子水平上受到调节。一个重要的 第一步是确定引发的酶促机制 由 RNA 聚合酶 II 和一组“起始因子”（称为 a、b、d、e 和 g) 从酵母中纯化。这些蛋白质现在可用于 数量足以进行机械研究。 因子 b 无法支持用纯形式重构的转录 聚合酶和其他起始因子，但因子 b“全酶” 具有该能力的已被隔离。同样，“天然”聚合酶- g因子复合物的比活性远高于 已鉴定出单独纯化的聚合酶或因子g。这些 全酶将被纯化以建立一个完全定义的系统 高效模板利用。 引发机制将通过物理和 酶学研究。蛋白质-DNA的结构和动力学 组装将通过非变性凝胶电泳确定 化学或酶“足迹”方法。单轮和多轮 转录方案将用于测试是否磷酸化和 RNA 聚合酶 II 的去磷酸化是反应的主要决定因素 效率。 因子 a，人类起始因子 TFIIE 的酵母同源物，将 从大肠杆菌中进行诱变和纯化以确定是否保守结构域 其一级结构对于转录活性很重要。 将分离并分析温度敏感突变体以确定 如果体内 RNA 合成需要因子 a/TFIIE。