title: 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-)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 step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase 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.
authors.affiliation: Institut für Biochemie, Freie Universität Berlin, FRG.
authors.forename: W D
authors.initials: WD
authors.lastname: Hardt
authors.forename: J
authors.initials: J
authors.lastname: Schlegl
authors.forename: V A
authors.initials: VA
authors.lastname: Erdmann
authors.forename: R K
authors.initials: RK
authors.lastname: Hartmann
mesh_terms: D001483:Base Sequence; D004722:Endoribonucleases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D014644:Genetic Variation; D006868:Hydrolysis; D007700:Kinetics; D007854:Lead / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012353:RNA, Transfer, Gly / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012353:RNA, Transfer, Gly; D007854:Lead; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium