Our goal is to understand the mechanism of transcription and its regulation. Determining the structure/function relationship of RNA polymerase, the enzyme responsible for RNA synthesis, is an essential step towards this goal. This is best accomplished with the highly characterized E. coli RNA polymerase. Because of the large size and complexity, this will require a combination of biochemical and biophysical methods to identify and characterize domains of the RNAP subunits, X-ray crystallography to determine high-resolution structures of RNA polymerase subunit domains, and electron microscopy to determine structures of intact assemblies. Many lines of evidence from our own work an the work of others indicate that RNA polymerase is modular in its architecture. The E. coli RNA polymerase is comprised of individual polypeptide subunits with stoichiometry alpha2betabeta'sigmna. The individual subunits, however, seem to be constructed of independent subdomains that have partial functions of their own. The goal of this proposal is to identify and characterize the subdomains that comprise the RNA polymerase subunits. More specifically, in terms of each subunit: 1) beta Subunit. We have already begun to elucidate the modular architecture of beta with a novel assay of in vitro assembly and function of RNA polymerase using fragments of beta. Using the same approach, we will further define the beta subunit modules. Detailed functional studies will address the roles of each module in; 1) the interaction with alpha in RNA polymerase assembly, 2) substrate nucleotide binding, and 3) transcript binding and modification. Detailed analysis of proteolytic sites with beta will be used to structurally characterize the beta subunit modules. 2) beta' Subunit. We will use the same approaches to dissect the beta; subunit into its modular components. Functional studies will address the roles of the beta' modules in the interaction with alpha2beta in RNAP assembly. Protoeolytic studies will be used to structurally characterize the beta' subunit modules. 3) sigma70 Subunit. We have undertaken studies to define and structurally characterize sigma70 domains using proteolysis. We will determine the X- ray crystal structure from crystals of a sub-domain containing conserved region 2, which interacts with the -10 promoter consensus sequence. Functional studies will address the roles of sigma70 domains in; 1) binding to core RNAP and binding and/or melting of specific promoter elements. 4) alpha Subunit. The domain architecture of the alpha subunit has been elucidated by others. We will undertake detailed structural studies of alpha subunit domains using the method of X-ray crystallography.
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