STRUCTURE AND FUNCTION OF RNA POLYMERASE IN E COLI

RNA聚合酶是基因表达的主要酶，也是基因表达的靶点。 基因调控 它的基本结构功能特征是高度 在所有生物体中都是保守的。该项目的总体目标是 对RNA聚合酶功能的分子机制的理解 和调控结构的分子遗传学方法， approach.为此，可以使用单个氨基酸取代、插入和修饰。 将在克隆的基因rpoB和rpoC中产生缺失，所述基因编码 RNA聚合酶的两个最大的亚基。突变亚基及其 然后，片段将从rpo表达质粒中过表达， 突变酶将从细胞中分离或从个体中重构 亚基及其片段。RNA的故障 由突变引起的聚合酶将使用一个武器库来表征， 允许监测和区分 RNA聚合酶功能循环的各个步骤。特别强调 将给予显性致死突变体的分析， RNA聚合酶的基本功能预计这一方法将 提供启动子结合、催化反应和 RNA的延伸和终止机制。的实验 缺失和亚基片段有望解剖两个大的 将RNA聚合酶的亚基转化为单独的结构域。这些结果将 有助于理解RNA聚合酶的一般结构， 成为直接结构研究的重要资源。测试 突变聚合酶在体外检测转录调控将有助于 确定调节因子的靶位点。实验也 提出了RNA聚合酶与抗生素利福平的相互作用 和具有直接公共卫生意义的链霉脂素。

RNA polymerase is the principal enzyme of gene expression and a 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 function and regulation in relation to structure using the molecular genetic approach. To this end, single amino acid substitutions, insertions and deletions will be generated in the cloned genes rpoB and rpoC which encode the two largest subunits of RNA polymerase. The mutant subunits and their fragments will then be overexpressed from rpo expression plasmids, and the mutant enzyme will be isolated from cells or reconstituted from individual subunits and their fragments in vitro. The malfunctioning of RNA polymerase caused by the mutations will be characterized using an arsenal of in vitro assays which allow to monitor and discriminate between individual steps of RNA polymerase functional cycle. Particular emphasis will be given to the analysis of dominant lethal mutants which disrupt vital basic functions of RNA polymerase. This approach is anticipated to provide molecular details of promoter binding, the catalytic reaction and the mechanism of elongation and termination of RNA. The experiments with deletions and subunit fragments are expected to dissect the two large subunits of RNA polymerase into individual domains. These results will help understanding of the general architecture of RNA polymerase and will become an important resource for direct structural studies. Testing of the mutant polymerase in in vitro assays of regulated transcription will help identify target sites for regulatory factors. Experiments are also proposed on the interaction of RNA polymerase with antibiotics rifampicin and streptolidigin which have direct public health significance.