KINETICS AND THERMODYNAMICS OF THE RNASE-P RNA CLEAVAGE REACTION - ANALYSIS OF TRANSFER-RNA 3'-END VARIANTS

We have studied the interaction of 3'-end variants of a (pre-)tRNA(Gly ) 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 tRNA(Gly) varia nts to RNase P RNAs was studied by 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 th e dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNA( Gly) variants. For E, coli RNase P RNA the following results were obta ined. Extensions of CCA by pCp or three nucleotides (AUA) stabilized g el-resolved tRNA(Gly) 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 b inding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy However, single turnover rate constants were only slig htly affected, indicating that a disruption or loss of the tRNA 3'-end -mediated interaction with RNase P RNA does not preferentially destabi lize the transition state. Our data suggest another kinetic step follo wing initial substrate binding to E. coli RNase P RNA (possibly a conf ormational rearrangement). For T. thermophilus RNase P RNA, product re lease of wild-type tRNA(Gly) CCAAUA was not rate-limiting in the multi ple turnover reaction. However, the effects of CCA mutations were simi lar to those attained with E. coli RNase P RNA. This supports the noti on that a high-affinity binding site for the tRNA 3'-end is a ubiquito us feature of eubacterial P RNAs. Finally, the results obtained here p rovide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of R Nase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.