The long-range goal of this project is to characterize the manner in which the RNA polymerases of bacteriophages T3 and T7 interact with transcription signals in the DNA template and perform their catalytic functions. Because of their structural simplicity and high degree of specificity, the phage polymerases are uniquely well suited for studies at the molecular level. Each of the phage enzymes consists of a single species of protein of ca 100 kd. Furthermore, the genes that encode the polymerases have been cloned and expressed in bacteria, making possible a variety of methods to analyze their structure and function. The specific goals are as follows: 1) Characterize the important structural features of the T3 and T7 promoters. The T3 and T7 promoters are both related to a highly conserved 23 bp consensus sequence. By examining the properties of synthetic mutant promoters, the elements of promoter structure that are required for binding and initiation will be examined. Similarly, the effects of methylating and ethylating promoters prior to polymerase binding will reveal important contacts between he enzyme and the DNA. 2) Identify functional domains within the enzyme. The T3 and T7 enzymes are highly specific for their own promoters. By constructing hybrid T3/T7 polymerase genes in vitro an 80 aa region of the polymerase that is responsible for specific promoter recognition has been identified. Site directed mutagenesis of the cloned polymerase genes will be used to characterize this region. In a parallel approach, the T3 and T7 RNA polymerase genes have been placed in plasmid and bacteriophage lambda vectors that allow the selection and characterization of polymerase mutants that are altered in transcription specificity, termination efficiency, and ability to participate in non-transcription activities such as replication and packaging of phage DNA. The active site of the polymerase may be specifically labelled through the use of reactive ribonucleotide derivatives; biochemical and genetic methods will be used to localize and characterize this region of the polymerase. Because all RNA polymerases carry out essentially the same series of steps in their reaction pathways, the information that we gain from these studies will serve to guide future experiments in more complex systems.
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