RNA SYNTHESIS BY BACTERIAL RNA POLYMERASE

拟议研究的目的是获得关于 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 针对不同亚基上决定簇的单克隆抗体 和完整的RNA聚合酶。 希望这样的收藏能够 含有序列和/或构象 依赖于RNA聚合酶的抗原决定簇。 在化学和酶促裂解后，抗原性结合位点被去除。 在可能的情况下，将通过有限的N-末端 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中的哪些区域 结构的表面上的自由和聚合酶相关的 亚单位。 单克隆抗体（推定）效应的相关性 对亚基-亚基组装、sigma、rho和cAMP受体蛋白介导的 影响，抑制聚合酶反应中的步骤，以及可能的 温度敏感性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 重组过程中RNA聚合酶的构象变化 启动子结合和三元延伸的形成 复合物可以使用那些单克隆抗体来研究， 决定因素受到这种复合物的影响。 亲缘关系 在不同细菌RNA聚合酶的抗原决定簇中 物种也将被调查。 化学物质的影响研究 重组和聚合酶活性的α亚基修饰 将继续进行。

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.