Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants

Journal of Molecular Biology
W D HardtR K Hartmann

Abstract

We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic st...Continue Reading

Citations

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