The long range goal of this project is to characterize the structure and function of bacteriophage T7 RNA polymerase (RNAP). The T7 enzyme is particularly attractive for such studies, as it consists of a single subunit of 883 amino acids that is capable of accurate transcription in the absence of any additional factors. Further, the phage RNAP exhibits homologies to other single subunit nucleotide polymerases with quite different template and substrate requirements, such as DNA polymerases and reverse transcriptases. Thus, characterization of the phage RNAP will contribute to our understanding of these enzymes as well. Four major areas of effort are planned: 1. Elements in the RNAP that are involved in promoter recognition will be characterized by selection of RNAP with altered specificities, and by directed mutagenesis of key regions of the RNAP. 2. The process of termination will be explored by genetic manipulation of signals that are recognized by T7 RNAP, selection of RNAP mutants that exhibit altered termination properties, and the identification of an RNA product binding site through the use of photoreactive substrate analogs. 3. Functional domains in the RNAP will be identified by characterization of mutant RNAP, using biochemical assays designed to assess their ability to carry out sequential steps in the transcription process. 4. The ability of the RNAP to transcribe DNA or RNA templates with novel topological structures will be examined.
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