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节。一个重要 第一步将是确定酶促引发机制 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。这些蛋白质现在可以在 数量足以进行机械研究。 因子B不能支持用纯形式的 聚合酶和其他起始因子，但因子B“全酶” 已经被隔离了同样，一种“天然”聚合酶- g因子复合物的比活性远高于 已经鉴定了分别纯化的聚合酶或G因子。这些 全酶将被纯化以建立一个完全确定的系统， 有效的模板利用率。 将通过物理和化学方法研究其引发机理， 酶学研究蛋白质-DNA的结构和动力学 将通过非变性凝胶电泳测定组装物， 化学或酶的“足迹”方法。单轮和多轮 转录方案将用于测试磷酸化和 RNA聚合酶II的去磷酸化是反应的主要决定因素 效率 因子a是人起始因子TFIIE的酵母同源物， 从E.以确定保守结构域 在其一级结构中对于转录活性是重要的。 温度敏感的突变体将被分离和分析，以确定 如果体内RNA合成需要因子a/TFIIE。

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.