RNA SYNTHESIS BY BACTERIAL RNA POLYMERASE

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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.