The precise regulation of transcription is central to cellular gene expression and genomic regulation. The long term goal of this research is to understand the enzymology of transcription, including the chemical basis of promoter recognition and the initiation of transcription. The family of small RNA polymerases from the T7 related bacteriophages presents an ideal model system in which to study, not only the fundamental aspects of sequence-specific recognition of DNA in transcription, but also the basic mechanisms of polymerases in general. Toward the latter end, this family of simple polymerases shows sequence homology to a large family of DNA and RNA dependent polymerases, including replicases from polio, influenza, and Sendai viruses and reverse transcriptases from hepatitis B and human immunodeficiency viruses. The simplicity of the T7 model system lends itself well to studies directed at understanding how individual protein-DNA functional group interactions lead to specific recognition of DNA and to the initiation of catalysis at a well-defined site in the DNA. The overall aim of this proposal is to further refine an emerging structural model for promoter recognition and to extend that model to include mechanistic detail regarding the early stages of transcription. The combination of powerful kinetic and thermodynamic assays with the direct incorporation of functional group substitutions into oligonucleotide based DNA templates allows detailed probing of critical protein-DNA interactions. A spectroscopic probe will be employed to monitor the thermodynamics and kinetics of local helix melting by the RNA polymerase, while quench-flow studies will be used to follow limited-turnover synthesis of RNA. Following the development of a mechanistic proposal for initiation, specific perturbations will be introduced into the promoter to provide a link between structure and function. The nature of the rate limiting step(s) in initiation will also be perturbed by alteration of the reaction conditions, to allow probes of various mechanistic steps. The precise role of the template strand in positioning substrate at the active site will be explored via structural modifications of the promoter DNA. Finally, modifications in the promoter will be selected for their ability to increase the processivity of transcription during incorporation of the first few ribonucleotides, in order to understand relationships between promoter release and abortive cycling.
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