CATALYTIC MECHANISM OF THE HAIRPIN RIBOZYME

Scripps Research Institute, La Jolla CA

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Abstract

DESCRIPTION: The long term goal of the work is to elucidate mechanisms of catalysis by small RNA enzymes. Detailed analyses of intermolecular hairpin an hammerhead ribozyme reactions revealed that the two catalytic RNAs use distinc kinetic mechanisms, the most striking difference being the propensity of the hairpin ribozyme to catalyze RNA ligation. pH and divalent metal cation independence of cleavage and ligation kinetics also point to a unique catalyti mechanism for the hairpin ribozyme. The investigator proposes to develop and test a detailed model of hairpin ribozyme catalytic chemistry. To ask if the highest energy barrier to hairpin cleavage is breaking the bond between phosphorus and the 5' oxygen, the investigator will determine cleavage rate constants for a hairpin substrate in which the reactive phosphodiester has bee replaced with a 5'-bridging phosphorothioate ester. Because sulfur is a better leaving group than oxygen, sulfur substitution of the bridging 5' oxygen is expected to accelerate phosphorus-sulfur bond breaking. If this step is rate-determining, cleavage of the thioester will be faster than cleavage of th natural substrate. Because a role for metal hydroxide as the general base in hairpin catalysis has been excluded, the investigator will examine functional groups in the ribozyme for the ability to catalyze proton transfer. Heteronuclear NMR experiments will be used to identify functional groups that might exhibit shifted pK values. Enhanced sensitivity to covalent modification by electrophilic reagents may reveal a potential general base catalyst within the hairpin ribozyme RNA. Nucleotide bases that might act as general acid or base catalysts will be replaced by base analogs with altered pKa values. Chang in the pH dependence of catalytic rate constants that results from the introduction of a modified nucleotide base with a shifted pKa value would provide strong evidence for the role of that base in catalytic chemistry. The investigator will attempt to identify ground state interactions that reflect a unusual ionization state of a nucleotide base. Finally, the investigator will explore why the hairpin ribozyme is a better ligase than it is a nuclease by determining the thermodynamics parameters of the internal equilibrium between cleavage and ligation and by assessing the role of tertiary interactions in stabilizing the ribozyme-substract complex.