RNA SYNTHESIS BY BACTERIAL RNA POLYMERASE

拟议研究的目的是获得更多关于 RNA聚合酶的结构与功能性质的关系 在很大程度上，这是一种免疫学方法。我们将生成一个库 针对不同亚基上的决定簇产生的单抗 和完整的RNA聚合酶。希望这样的收藏品将 含有序列和/或构象的单抗 依赖于RNA聚合酶的抗原决定簇。 在化学和酶作用下切割抗原的位置 决定因素将在可能的情况下由有限的N-末端确定 多肽片段的测序。抗原区的定位 将指示天然折叠的多肽链的哪些区域 表面的结构是游离的和聚合酶相关的 亚单位。单抗(假定)效应的相关性 亚基-亚基组装、Sigma、Rho和cAMP受体蛋白介导 聚合酶反应中步骤的影响，抑制，以及可能的 温度敏感型聚合酶突变体对细胞的保护作用 抗原区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 亚基、启动子结合和三元延伸的形成 可以用其抗原性的单抗研究复合体。 决定因素受到这样的复合体的影响。系统发育关系 不同细菌RNA聚合酶抗原决定簇的比较 还将对物种进行调查。关于化学药剂作用的研究 α亚基对重组和聚合酶活性的修饰 将会继续。

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.