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.
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