Despite intensive analysis of hammerhead ribozyme biochemistry and the elucidation of two crystal structures, the active site structure and reaction mechanism remain unknown. In contrast, work in the hairpin ribozyme system has provided a much clearer view of active site assembly, and has led to the development and experimental verification of structural and mechanistic models. The proposed work is based on two testable hypotheses. First, Dr. Burke proposes that the existing crystal structures of the hammerhead ribozyme represent a state analogous to the undocked structure of the hairpin ribozyme, and that a significant conformational change analogous to hairpin ribozyme docking is an essential step in assembly of the hammerhead active site, and is an obligatory prelude to cleavage. Second, recent experimental work on the hammerhead system in Dr. Burke's laboratory leads him to propose that the hammerhead and hairpin ribozymes share common features of active site structure and reaction mechanism, and that G12 of the hammerhead ribozyme may be structurally and functionally analogous to G8, a key catalytic base in hairpin ribozyme catalysis.
Specific Aims of the proposal are: (1) Identify conformational changes required to assemble the active site, (2) Identify essential components of the hammerhead active site, and (3) Develop and test models for active site structure and mechanism. In these studies, the extensive repertoire of experimental methods that Dr. Burke and his group have developed for their studies of hairpin ribozymes will be extremely valuable. The results will provide critical insights into the molecular evolution of biological catalysts and the mechanistic strategies employed by RNA and ribonucleoprotein enzymes, and will directly contribute to the use of ribozymes in functional genomics and gene therapy.
