Kinetics and Thermodynamics of the RNase P RNA Cleavage Reaction:Analysis of tRNA 3'-end Variants

Abstract

We have studied the interaction of 3′-end variants of a (pre-)tRNAGlywith ribonuclease P (RNase P) RNAs fromEscherichia coliandThermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGlyvariants to RNase P RNAs was studied bu gel retardation. A newly developed assay, based on the reduction of Pb2+-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-tRNAGlyvariants. ForE. coliRNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGlybinding 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 step following initial substrate binding toE. coliRNase P RNS (possibly a conformational rearrangement. ForT. thermo philusRNase P RNA, product release of wild-type tRNAGlyCCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained withE. coliRNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3′-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.