The main goal of the project is to understand how specific RNA structures contribute to RNA catalysis. The major experimental system will continue to be the self-splicing IVS (intervening sequence) of Tetrahymena and ribozymes (RNA enzymes) derived from the IVS. Additional goals are to develop a set of sequence- specific RNA cleavage enzymes that may be useful as tools for RNA molecular biology and to explore the generality of RNA catalysis in new directions.
Specific aims are the following: (1) Obtain a 3-dimensional view of the active site of the Tetrahymena IVS RNA with and without the substrates bound. Approaches will include chemical modification, cleavage of the RNA by an active-site-directed inhibitor, and UV crosslinking. (2) Identify nucleotides involved in substrate- binding and catalysis using site-specific and random mutagenesis. A particular model of the 3-dimensional structure of the catalytic core will be tested. (3) Further explore the activity of the ribozyme as a sequence-specific endoribonuclease. Twenty active- site variants with altered substrate specificity will be characterized. (4) Test the idea that nuclear mRNA splicing is at least in part catalyzed by small nuclear RNAs using the Saccharomyces cerevisiae system. (5) Test the idea that mRNA stability might in some cases be determined by the presence of self-cleavage sites. The Tetrahymena IVS RNA provides an unusually amenable system for learning about structure-function relationships in RNA and biological catalysis in general. It is likely that many of he findings will be applicable to other systems where RNA is in catalysis, including other RNA processing reactions and protein synthesis.
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