BIOCHEMISTRY OF SPECIFIC INITIATION BY RNA POLYMERASE II

这项研究的最终目标是了解真核生物 信使RNA的合成在分子水平上受到调控。一个重要的 第一步是确定起始酶的机制。 由RNA聚合酶II和一系列“起始因子”(称为 A、b、d、e和g)从酵母中纯化。这些蛋白质现在可以在 足以进行机械学研究的量。 B因子不支持纯种形式重组的转录 聚合酶和其他启动因子，但a因子b“全酶” 拥有这种能力的人已经被孤立了。同样，一种“天然”聚合酶- 比活性高得多的g因子复合体 已鉴定出单独纯化的聚合酶或g因子。这些 全酶将被提纯以建立一个完全定义的系统 有效地利用模板。 引爆机理将通过物理和化学方法进行研究。 酶学研究。蛋白质-DNA的结构和动力学 组件将通过非变性凝胶电泳法和 化学或酶的“足迹”方法。单发和多发 转录方案将被用来测试磷酸化和 RNA聚合酶II的去磷酸化是反应的主要决定因素 效率。 A因子，人类启动因子TFIIE的酵母同系物，将 从大肠杆菌中诱变和纯化以确定保守结构域是否 在其一级结构中对转录活性很重要。 对温度敏感的突变体将被分离和分析，以确定 如果在体内合成RNA需要a/TFIIE因子。

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.