The relationship between RNA structure and function will be studied using two RNA catalyzed reactions, the hammerhead ribozyme and the ribosome. These two RNA catalysts differ dramatically in size and catalyze quite different reactions, but show similar rate enhancements over the uncatalyzed reaction. Kinetically well behaved versions of hammerheads containing a newly discovered tertiary interaction will be used to evaluate the mechanism of this small ribozyme. Experiments will test whether the catalytically active form of the hammerhead requires a transient docking within its catalytic core to form a structure that differs substantially from the current X-ray structure. A newly developed assay, that measures the ability of the E. coli ribosome to protect the labile aminoacyl linkage from hydrolysis or aminolysis, will be exploited as an independent means to evaluate accessibility and positioning of the ester linkage within the ribosome active site. In addition, two new assays will be developed to measure the rate of peptide bond formation by the E. coli ribosome using intact aminoacyl and peptidyl tRNA substrates. These will avoid the artifacts encountered when incomplete substrates are used. The hydrolysis protection and peptidyl transferase assays will be used to examine the consequences of structural perturbations on the ribosomal active site. These experiments will help resolve several unanswered questions on the mechanism of peptide bond formation by the ribosome. ? ? ?
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