The overall goal of this project is to characterize the transcription process mediated by bacteriophage T7 RNA polymerase (RNAP) and, where possible to interpret this process with regard to the structure of the enzyme as determined by crystallographic methods. Although the phage RNAP consists of a single subunit, it is able to perform all the steps in the transcription cycle that are carried out by the more complex multisubunit RNAPs found in prokaryotic and eukaryotic cells. Furthermore, T7 RNAP is related to members of a superfamily of nucleotide polymerases that include DNA-directed DNA polymerases, mitochondrial RNAPs, and RNA-directed polymerases (including reverse transcriptases). Studies of this enzyme will therefore contribute to our understanding of nucleotide polymerization in general, and DNA-dependent RNA synthesis in particular.
The specific aims of the proposal are: 1) To study transcript termination at a novel class of signals that involves recognition of a conserved 7 bp sequence. These experiments will involve biochemical and kinetic analyses, and the selection of mutant RNAPs that can recognize altered signals. 2) To study promoter recognition and function by constructing and analyzing mutant RNAPs in which the specificity loop (a structural element that is required for promoter recognition) is altered. 3) To characterize interactions between the template DNA, and the nascent RNA, and the RNAP by cross-linking methods, and to localize these interactions within the structure of the enzyme. These methods will be applied to complexes formed at a number of stages in the transcription process. 4) To define structural elements in the transcription complex that are involved in initiation and stabilization of the elongation complex through the use of synthetic templates with altered topologies.
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