STRUCTURE AND FUNCTION OF RNA POLYMERASE IN E. COLI

RNA聚合酶是基因表达的主要酶，而 基因调控的目标。其基本结构--功能 这些特征在所有活着的有机体中都高度保守。这个 这个项目的主要目标是理解分子 利用定点定位研究RNA聚合酶的基本功能机制 进化不变氨基酸在个体中的突变 大肠埃希菌RNA聚合酶亚基。为了这个 目的，单一氨基酸替代突变，连接子 插入和框内删除将在克隆的 编码核心RNA聚合酶β亚基的基因rpoB。这个 突变的RNA聚合酶全酶(α-2-β-β-‘sigma)将 然后在体外为生化表征做准备 从单个亚基重组而来。这些亚单位将是 通过从携带三种基因的质粒中过度表达它们而获得 可诱导启动子控制下的RPO基因。这个 氨基酸引起的RNA聚合酶功能障碍 替代将使用体外武器库进行表征。 允许监测和量化各个步骤的分析 这种酶的功能循环。在这个过程的第二阶段 项目，补偿抑制突变将被分离并 已映射。此方法预计将提供有关以下方面的信息 启动子结合的分子细节，启动子的功能 酶的催化中心，加工性的本质， 暂停和终止的机制，以及揭示 执行这些功能的酶的结构要素。

RNA polymerase is the principal enzyme of gene expression and the target for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. The broad goal of this project is the understanding of the molecular mechanism of RNA polymerase basic function using site directed mutagenesis of evolutionarily invariant amino acids in individual subunits of RNA polymerase from Escherichia coli. For this purpose, single amino acid substitution mutations, linker insertions and in-frame deletions will be generated in the cloned gene rpoB which encodes the core RNA polymerase beta subunit. The mutant RNA polymerase holoenzyme (alpha-2-beta-beta-'sigma) will then be prepared for biochemical characterization by in vitro reconstitution from the individual subunits. The subunits will be obtained by overexpressing them from plasmids carrying the three rpo genes under the control of inducible promoter. The malfunctioning of RNA polymerase caused by the amino acid substitutions will be characterized using an arsenal of in vitro assays which would allow to monitor and quantitate individual steps of the enzyme's functional cycle. At the second stage of this project, compensatory suppressor mutations will be isolated and mapped. This approach is anticipated to provide information about molecular details of promoter binding, the functioning of the enzyme's catalytic center, the nature of processivity, the mechanism of pausing and termination, as well as to, uncover structural elements of the enzyme which carry out these functions.