The overall goal of this project is to characterize fundamental aspects of the transcription process using bacteriophage T7 RNA polymerase (RNAP) as a model system, and to interpret this process with regard to the organization and structure of the enzyme. Although the phage RNAP consists of a single subunit, it carries out the same steps in the transcription cycle as the multisubunit RNAPs found in bacterial and eukaryotic cells. Furthermore, T7 RNAP is a member of a superfamily of nucleotide polymerases that includes DNA polymerases of the pol I type, mitochondrial RNAPs, and reverse transcriptases. Studies of the phage enzyme will therefore contribute to our understanding of RNA synthesis in the general context of nucleotide polymerization.
The specific aims of the application are to: Define the structural basis for the transition from an initiation complex (IC) to an elongation complex (EC). Like other RNAPs, T7 RNAP forms an unstable IC before it isomerizes to a stable EC. This process involves multiple steps and intermediate conformations. To examine this, transcription complexes arrested at various stages will be characterized by biochemical and structural analyses, and the functional importance of key structural elements will be examined by site-directed mutagenesis. Determine the functional importance of key structural elements in the elongation complex. Recent crystallographic analyses of a T7 TNAP EC have identified structural elements that are likely to be critical for complex stability and transcript elongation. The functional importance of these elements will be characterized by site-directed mutagenesis and by nucleic acid: RNAP crosslinking experiments. Characterize the termination process. It is thought that termination involves a reversal of the process that leads to a stable EC. The organization of transcription complexes that are halted just before the termination release site will be examined using photocrosslinking techniques and crystallographic analysis.
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