X-Ray Crystallographic Studies of N4 RNA Polymerases

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DESCRIPTION (provided by applicant): Transcription is the major control point of gene expression and RNA polymerase (RNAP) is the central target of regulation in this process. Our long-term goal is to understand the mechanisms and functions of RNAP and its regulation. In addition to biochemical and biophysical approaches, determining high-resolution three-dimensional structures is an essential step. I recently purified, crystallized, and solved the 2.0 Angstroms resolution X-ray crystal structure of the functional RNAP domain (1106 amino acid residues, mini-vRNAP) of bacteriophage N4-encoded virion-encapsulated RNAP (total 3,500 amino acid residues). This represents a major breakthrough in our work. Here, I propose further structural studies aimed towards increasing our understanding of RNAP function and the mechanism of transcription. Specific aims are: 1. Solve crystal structure of mini-vRNAP and promoter DNA binary complex. I have obtained promising crystals of the binary complex; 2. Structurally characterize the transcription initiation process. I have found conditions for preparing a transcription initiation complexes (binary complex with two substrate nucleotides in active site); 3. Solve crystal structure of a transcription elongation complex. We will prepare mini-vRNAP transcription elongation complexes for crystallization and solve the structure. 4. Solve the crystal structure of N4 RNAPII. We will crystallize and solve the structures of N4 RNAPII and RNAPII complex with gp2, an essential cofactor. These studies will provide insights into the mechanism of transcription of the T7 bacteriophage-like single-subunit RNAP family, and specifically those enzymes in the family that require accessory factors.