9308670 Martin The specific aims of this proposal are to test and 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 incorporation of functional group substitutions in the protein allow detailed probing of critical protein-DNA interactions in 3 areas of the promoter: a proposed duplex recognition domain upstream, a single stranded region near the start site, and the poorly understood transition region in between. A variety of approaches will be applied to this system to complement well-established kinetic studies and to provide a better understanding of the chemical nature of specific interactions. Titration microcalorimetry will be developed to probe protein - DNA binding interactions in more detail. Specific chemical incorporation of nonperturbing photoactivatable nucleotides into promoter DNA will provide much needed direction to further protein mutagenesis studies. %%%% The goal of this research is to understand the enzymology of transcription, including the chemical basis of transcript initiation. The T7 family of RNA polymerses present an ideal model system in which to study not only the fundamental aspects of sequence-specific macromolecular interactions, but also the basic mechanisms of polymerases. The effects of simple chemical modifications in the DNA and the protein will be determined, in order to refine our understanding of the recognition interface. ****
