Structure and Function of RNA Polymerases in E Coli

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DESCRIPTION (provided by applicant): RNA polymerase (RNAP) is the principal enzyme of gene expression and the target for genetic regulation. The long-term objective of this research is the understanding of the function of bacterial RNAP as a molecular machine at the atomic level of resolution. Specifically, the aims are (1) to build a model of the active center that would assign function to specific amino acid residues, nucleotides and metal ions in reactions of RNA synthesis and degradation and to interpret structurally the interrelationship between these reactions; (2) to characterize conformational transitions in the ternary transcribing complex that modulate catalytic function; (3) to understand transitions in the initial transcribing complex that take place during initial buildup of the nascent transcript, the release of the initiation factor sigma and promoter clearance; and (4) to explore the plasticity of RNAP molecule through generation of aptamers so that multiple conformations of RNAP could be captured for crystallographic studies. To these ends, a series of functionally defined complexes will be generated, in which RNAP will be (a) stalled at a particular stage of the transcription process; or (b) complexed with a defined nucleic acid scaffold, or (c) frozen in a complex with a bound aptamer. The complexes will be studied using chemical nucleic acid-protein crosslinks, genetically engineered mutations in RNAP, and discriminative biochemical assays. The results will be interpreted with the aid of molecular modeling. The understanding of the basic transcription mechanisms generated in this research is a prerequisite for molecular interpretation of regulatory phenomena and the development of interventions into gene expression. In addition, knowledge gained in these studies will be helpful for rational design and screening of new inhibitors of RNAP, which has been a proven target for anti-microbial therapy.