RNA SYNTHESIS BY BACTERIAL RNA POLYMERASE

通过细菌 RNA 聚合酶合成 RNA

• 批准号: 3269345
• 负责人: JOSEPH S KRAKOW
• 金额: $ 14.9万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-11-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3269345
• 关键词: Azotobacter vinelandii; Escherichia coli; RNA directed DNA polymerase; antibody formation; bacterial RNA; chemical models; conformation; enzyme induction /repression; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; genetic library; genetic mapping; high performance liquid chromatography; molecular site; monoclonal antibody; nucleic acid reconstitution; nucleic acid sequence; radiotracer

The purpose of the proposed research is to gain further information on the relation of structure to the functional properties of RNA polymerase using for the most part an immunological approach. We will generate a library of monoclonal antibodies raised against determinants on the separate subunits and the intact RNA polymerase. It is hoped that such a collection would contain monoclonal antibodies which were sequence and/or conformation dependent with regard to antigenic determinants on RNA polymerase. Following chemical and enzymatic cleavage the sites of the antigenic determinants will be determined where possible by limited N-terminal sequencing of the polypeptide fragments. Mapping of the antigenic domains will indicate which areas of the polypeptide chains in the native folded structure are on the surface of the free and polymerase-associated subunits. Correlation of the (putative) effects of monoclonal antibodies on subunit-subunit assembly, sigma, rho and cAMP receptor protein mediated effects, inhibition of steps in the polymerase reaction, and possible protective effects with temperature-sensitive polymerase mutants with the antigenic domains can relate enzyme structure with function. The probable conformational changes in RNA polymerase during reconstitution from subunits, promoter binding and formation of the ternary elongation complexes can be studied using those monoclonal antibodies whose antigenic determinants are affected by such complexes. The phylogenetic relationship among the antigenic determinants of RNA polymerase from different bacterial species will also be investigated. Studies on the effect of chemical modification of the alpha subunit on reconstitution and polymerase activity will be continued.

拟议研究的目的是获得有关 RNA聚合酶的结构与功能特性的关系 大部分是免疫学方法。 我们将生成一个库 针对单独亚基上的决定簇产生的单克隆抗体 和完整的RNA聚合酶。 希望这样的合集能够 含有序列和/或构象的单克隆抗体 依赖于RNA聚合酶的抗原决定簇。 化学和酶促裂解抗原位点后 决定因素将尽可能由有限的 N 端决定 多肽片段的测序。 抗原结构域的定位 将指示天然折叠的多肽链的哪些区域 结构位于游离和聚合酶相关的表面 亚单位。 单克隆抗体（推定）效应的相关性 亚基-亚基组装、sigma、rho 和 cAMP 受体蛋白介导 效应、聚合酶反应步骤的抑制以及可能的 温度敏感聚合酶突变体的保护作用 抗原结构域可以将酶的结构与功能联系起来。 可能的 RNA聚合酶在重组过程中的构象变化 亚基、启动子结合和三元延伸的形成 可以使用那些具有抗原性的单克隆抗体来研究复合物 决定因素受此类复合物的影响。 系统发育关系 来自不同细菌的RNA聚合酶的抗原决定簇 物种也将受到调查。 化学作用的研究 α亚基的修饰对重构和聚合酶活性的影响 将继续。