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: 2833522
• 负责人: KONSTANTIN V SEVERINOV
• 金额: $ 18万
• 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/2833522
• 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其分子机制 DNA依赖性RNA聚合酶（RNAP）的转录和结构—— 转录的主要酶和主要靶标 规定。 细胞 RNAP 是多功能、多亚基分子 机器。 分离的RNAP亚基不具有部分生化特性 酶的功能（例如，解链 DNA 或结合 NTP 的能力） 基材）。 因此，RNAP 功能位点可能是通过变构变化形成的 或在完整酶组装后的亚基界面处。 了解酶过程中亚基间和亚基内的相互作用 组装可以为 RNAP 结构提供重要的关键 转录机制。 我们将继续检查装配和 大肠杆菌 RNAP 的结构（亚基组成 alpha2betabeta')，同类中最了解的酶，也是 我们最近获得了胃病原菌幽门螺杆菌的 RNAP 显示包含两个最大亚基的最不寻常的自然融合 （β-β）。 这将需要： 在大肠杆菌中——抑制子分析 条件RNAP组装突变体；交互域的识别 使用酵母双杂交系统，并对组装进行生化分析 由 RNAP 亚基片段以及金属形成的中间体 取代、原子吸收光谱、局域自由基 足迹和荧光能量转移测量来定位 对酶组装很重要的两个 RNAP 锌离子中的每一个；在 螺杆菌-rpoB（β）-rpoC（β'）基因的DNA序列分析 幽门螺杆菌以外的螺杆菌种中的组织，以及 相关属，例如弓形杆菌属；工程化幽门螺杆菌菌株 分离 rpoB 和 rpoC 基因，测试该菌株的活力和 在培养物和适当的动物模型中的生长活力， 幽门螺杆菌 RNAP 的纯化和/或体外重建，以及 在确定的启动子上建立体外转录系统， 以及寻找监管因素。 各研究结果 RNAP 应该极大地丰富我们与其他研究的共同研究 导致对真细菌 RNAP 组装的新的重要见解， 结构、功能和生物调节。 因为坚强 RNAP 的进化保守性，这项工作也与 真核转录，其中所提出的分析尚未完成 可行的。 该项目的大部分健康相关性源自 它对理解基本转录机制的贡献 胃部病原体幽门螺杆菌和相关致病菌。