STRUCTURE AND FUNCTION OF RNA POLYMERASE IN E. COLI

RNA聚合酶是基因表达的主要酶， 基因调控的目标。 其基本结构-功能 特征在所有生物体中高度保守。 的 该项目的主要目标是了解分子 RNA聚合酶基本功能的机制 个体中进化不变氨基酸的诱变 来自大肠杆菌的RNA聚合酶的亚基。 为此 目的，单氨基酸取代突变，接头 插入和框内删除将在克隆的 基因rpoB，其编码核心RNA聚合酶β亚基。 的 突变RNA聚合酶全酶（α-2-β-β-σ）将 然后制备用于体外生物化学表征， 由单个亚基重组而成。 各分单位将 通过从携带三个 诱导型启动子控制下的rpo基因。 的 由氨基酸引起的RNA聚合酶的故障 取代将使用体外免疫组织化学的武库来表征。 允许监测和定量单个步骤的测定 酶的功能循环。 在第二阶段， 项目，补偿抑制突变将被隔离， 映射。 预计这种方法将提供以下信息： 启动子结合的分子细节， 酶的催化中心，持续合成能力的性质， 暂停和终止的机制，以及揭示 执行这些功能的酶的结构元件。

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.