Catalytic RNAs provide a window into a primordial 'RNA World' from which modern biology might have evolved. Contemporary roles for RNA catalysts in the modern protein world are still found in the regulation of gene expression and in protein synthesis, making RNA catalysis essential to normal growth and development. Recent high-resolution structures of self-cleaving and self-splicing RNAs provide pictures of the active sites for the hammerhead, hepatitis delta virus, and hairpin self-cleaving RNAs and Group-l self- splicing introns. The structure of the ribosome gave us a view of a peptidyl transferase center constructed entirely from RNA. Mechanistic studies carried out over more than 20 years since ribozymes were first discovered can now be compared and contrasted with these structures, laying the groundwork for experiments to probe fundamental questions about how RNA enzymes use their functional groups for catalysis. As the first ribozyme shown to function without metal cation cofactors, the hairpin ribozyme is a prototype for understanding metal-independent RNA catalysis. Work during previous award periods defined the essential structural and biochemical features of the active site and led to testable models for the roles of active site nucleobases in catalytic chemistry. Our goal for the coming award period is to define the catalytic mechanism of the hairpin ribozyme and elucidate the mechanisms of a recently discovered metabolite- dependent ribozyme, which resembles the hairpin ribozyme in certain key mechanistic features. Specific goals include 1) distinguishing contributions to bond breaking and bond making steps in the hairpin ribozyme reaction using 5' phosphorothiolate RNAs, 2) determining the microscopic pKa values of hairpin ribozyme active site nucleobases using 8-azapurine fluorescence to monitor pH-dependent changes in protonation states and using heteronuclear NMR to monitor pH-dependent changes in chemical shifts, 3) determine the energetic contributions of individual active site functional groups to ground state and transition state stability by combining atomic level mutagenesis with physical and functional assays of hairpin ribozyme complex formation and catalysis, and 4) establish a kinetic and thermodynamic framework for quantitative analysis of the reaction pathway of the metabolite-responsive glmS ribozyme. The proposed studies will generate fundamental insights into mechanisms of catalysis by RNA and RNP enzymes, RNA folding and dynamics, and RNA interactions with small ligands. This work will deepen our basic understanding of RNA structure and function, which is a critical part of the fundamental biology of health and disease. The results of these studies also will provide a framework to guide the development of technical and therapeutic applications involving RNAs as targets and reagents.
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