GENETICS AND BIOCHEMISTRY OF RNA POLYMERASE ASSEMBLY

RNA 聚合酶组装的遗传学和生物化学

• 批准号: 6636299
• 负责人: KONSTANTIN V SEVERINOV
• 金额: $ 28.96万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636299
• 关键词: DNA directed RNA polymerase; Escherichia coli; Helicobacter; atomic absorption spectrometry; bacterial genetics; bacterial proteins; chemical binding; chemical substitution; cobalt; enzyme biosynthesis; enzyme structure; fluorescence resonance energy transfer; gene mutation; genetic transcription; iron; protein purification; protein sequence; protein structure function; yeast two hybrid system; zinc

Our long-term goal is to understand the molecular mechanism of transcription and the structure of DNA-dependent RNA polymerase (RNAP)-- the principle enzyme of transcription, and a primary target of regulation. Cellular RNAPs are multifunctional, multisubunit molecular machines. Isolated RNAP subunits do not possess partial biochemical functions of the enzyme (e.g., the ability to melt DNA, or bind NTP substrates). Thus, RNAP functional sites may form by allosteric changes or at subunit interfaces upon assembly of complete enzyme. Understanding inter- and intrasubunit interactions during the enzyme assembly could provide an important key to RNAP structure and the mechanism of transcription. We will continue examining the assembly and structure of RNAP from Escherichia coli (subunit composition alpha2betabeta'), the best understood enzyme of its class, and also the RNAP of the gastric pathogen Helicobacter pylori, which we have recently shown contains a most unusual natural fusion of the two largest subunits (beta-beta ). This will entail: in E. coli--suppressor analysis of conditional RNAP assembly mutants; identification of interacting domains using the yeast two-hybrid system, and biochemical analysis of assembly intermediates formed with fragments of RNAP subunits, as well as metal substitution, atomic absorption spectroscopy, localized radical footprinting and fluorescence energy transfer measurements to localize each of the two RNAP zinc ions important for the enzyme assembly; in Helicobacter- DNA sequence analyses of rpoB(beta)-rpoC(beta') gene organization in Helicobacter species other than H. pylori, and in related genera such as Arcobacter; engineering an H. pylori strain with separate rpoB and rpoC genes, tests of this strain for viability and vigor of growth in culture and in appropriate animal models, purification and/or in vitro reconstitution of H. pylori RNAP, and establishment of an in vitro transcription system on defined promoters, and the search for regulatory factors. The results of studies of each RNAP should greatly enrich our studies with the other, and collectively lead to new important insights into eubacterial RNAP assembly, structure, function and biological regulation. Because of strong evolutionary conservation of RNAP, this work is also highly relevant to eukaryotic transcription, where the proposed analyses are not yet feasible. Much of the health relatedness of this project derives from its contribution to the understanding of basic transcription machinery in gastric pathogen H. pylori, and related pathogenic bacteria.

我们的长期目标是了解 转录和DNA依赖性RNA聚合酶（RNAP）的结构 - 转录的原理酶，也是 规定。 细胞RNAP是多功能的多亚基分子 机器。 孤立的RNAP亚基不具有部分生化 酶的功能（例如，融化DNA或结合NTP的能力 基材）。 因此，RNAP功能位点可能通过变构变化形成 或在组装完整酶时在亚基界面处。 了解酶期间的亚氨间相互作用 组装可以为RNAP结构提供重要的关键， 转录机理。 我们将继续研究大会， 大肠杆菌的RNAP结构（亚基组成 alpha2betabeta'），其班级的最佳理解酶，也是 胃病原体幽门螺杆菌的RNAP，我们最近有 所示包含两个最大亚基的最异常的自然融合 （beta-beta）。 这将需要：在E. coli-spressor分析中 有条件的RNAP组装突变体；识别相互作用域 使用酵母双杂交系统，以及组装的生化分析 由RNAP亚基的碎片和金属形成的中间体 取代，原子吸收光谱，局部自由基 足迹和荧光能量传递测量值 两个RNAP锌离子中的每一个对于酶组件很重要；在 RPOB（beta）-RPOC（beta'）基因的Helicobacter- DNA序列分析 幽门螺杆菌以外的其他螺旋杆菌中的组织 相关属，例如弧菌；工程幽门螺杆菌菌株 单独的RPOB和RPOC基因，该菌株的生存力和 文化和适当动物模型的增长活力， 幽门螺杆菌的纯化和/或体外重构，以及 在定义的启动子上建立体外转录系统， 以及寻找监管因素。 每个研究的结果 RNAP应该大大丰富我们的研究，并集体地 导致新的重要见解对细菌RNAP组装， 结构，功能和生物调节。 因为强大 RNAP的进化保护，这项工作也与 真核转录，尚未提出的分析尚未 可行的。 该项目的健康相关性大部分来自 它对理解基本转录机械的贡献 幽门螺杆菌和相关的致病细菌中。