The site-specific cleavage of double-stranded RNA by ribonuclease III of Escherichia coli is a key step in the maturation, function and decay of cellular and viral RNAs. Nucleases similar to RNase III occur in eukaryotic cells, and perform similar functional roles. The long-term objective is to determine the enzymatic mechanism of double-stranded RNA recognition and cleavage and its role in RNA maturation, function and decay. Specific Watson- Crick base-pair sequences determine the RNase III binding site, and an internal loop determines whether one or both RNA strands are cleaved The pattern of cleavage in turn controls RNA function and half-life.
The Specific Aims are to: 1. Determine the Watson-Crick base-pair sequence features which confer specific binding of RNase III. RNase III recognition of substrate is dependent upon specific Watson-Crick base-pair (W-C bp) sequences near the cleavage site. To determine W-C bp sequence features which confer specific binding, substrates containing bp substitutions or base analogues lacking functional groups will be tested for their in vitro binding and cleavage activities. In vitro genetic selection will determine the range of W-C bp sequences that confers specific binding. 2. Determine the RNA structural features that confer single-strand cleavage. An internal loop switches the pattern of double-strand cleavage to single-strand cleavage. To determine how this motif allows RNase III binding, but confers single-strand cleavage, mutant substrates with altered internal loop sequences will be tested for their binding affinities and cleavage reactivities. In vitro genetic selection will be used to determine the range of internal loop structures that confer binding and/or cleavage. 3. Determine 2'-hydroxyl group involvement in binding, and the energetic contribution of ionic and hydrophobic interactions. A specific set of substrate 2'-hydroxyls and phosphodiester oxygens may directly contact RNase III and contribute to overall binding energy. To identify the 2'-hydroxyl groups important for binding 2'-deoxyphosphorothioate-substituted RNAs will be used in modification-interference assays, and site-directed 2'-deoxy- substituted substrates will be tested for their in vitro binding and cleavage reactivities. The ionic and hydrophobic contributions to binding will be determined by measuring substrate binding affinities as a function of salt concentration and temperature, respectively.
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