The recognition and cleavage of double-helical RNA is essential for the maturation, function, and decay of cellular and viral RNA molecules. These processes are catalyzed by double-stranded(ds)-RNA-specific ribonucleases. The long-range goal of this project is to determine the enzymatic mechanism of dsRNA cleavage and its role in regulating cellular and viral RNA metabolism and function. The short-term goal is to analyze Escherichia coli ribonuclease III, a dsRNA-specific nuclease that participates in rRNA, tRNA and mRNA maturation, function and decay. RNase III contains a domain that specifically binds dsRNA, as well as a second, catalytic domain which can bind and site-specifically cut dsRNA. dsRNA-specific ribonucleases with similar domains are found in all cells examined, and perform similar functional roles. A model for RNase III action has been developed that involves the coordinated action of the dsRNA-binding and catalytic domains. To test and refine this model the specific aims are to: 1. Determine how the dsRNA-binding domain enhances substrate cleavage by the catalytic domain. Interactions between the catalytic domain, dsRNA-binding domain, and dsRNA substrate required for efficient cleavage will be identified by crosslinking, protection and interference assays on specific protein-dsRNA complexes. 2. Identify catalytic domain components essential for dsRNA cleavage. The conserved, charged or polar amino acids in the catalytic domain will be mutated and mutant enzyme cleavage activities measured by kinetic assays. The dsRNA-binding and cleavage activities of genetically-selected RNase III mutants (provided by collaborator R. W. Simons) also will be measured. The minimum number of metal ions required for the cleavage step will be determined by kinetic and equilibrium binding experiments. 3. Identify dsRNA-binding domain components directly involved in dsRNA recognition. Conserved residues in the putative recognition surface of the dsRNA-binding domain will be mutated and the dsRNA-binding and cleavage activities measured by gel mobility shift and filter binding assays. The dsRNA-binding activities of mutants obtained by genetic screens will be similarly measured. Mutations which disrupt functional interactions between the dsRNA-binding and catalytic domains will be identified and characterized.
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