Conformational change in the Bacillus subtilis RNase P holoenzyme--pre-tRNA complex enhances substrate affinity and limits cleavage rate.
Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the 5' maturation of precursor tRNAs. To investigate the mechanism of substrate recognition in this enzyme, we characterize the thermodynamics and kinetics of Bacillus subtilis pre-tRNA(Asp) binding to B. subtilis RNase P holoenzyme using fluorescence techniques. Time courses for fluorescein-labeled pre-tRNA binding to RNase P are biphasic in the presence of both Ca(II) and Mg(II), requiring a minimal two-step association mechanism. In the first step, the apparent bimolecular rate constant for pre-tRNA associating with RNase P has a value that is near the diffusion limit and is independent of the length of the pre-tRNA leader. Following formation of the initial enzyme-substrate complex, a unimolecular step enhances the overall affinity of pre-tRNA by eight- to 300-fold as the length of the leader sequence increases from 2 to 5 nucleotides. This increase in affinity is due to a decrease in the reverse rate constant for the conformational change that correlates with the formation of an optimal leader-protein interaction in the RNase P holoenzyme-pre-tRNA complex. Furthermore, the forward rate constant for the conformational change becomes rate limiting for cleavage under single-turnover conditions at high pH, explaining the origin of the observed apparent pK(a) in the RNase P-catalyzed cleavage reaction. These data suggest that a conformational change in the RNase P*pre-tRNA complex is coupled to the interactions between the 5' leader and P protein and aligns essential functional groups at the cleavage active site to enhance efficient cleavage of pre-tRNA.
Hsieh
John
J
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Fierke
Carol A
CA
D001412:Bacillus subtilis / Q000378:metabolism*; D019793:Fluorescein; D005456:Fluorescent Dyes; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D020035:Holoenzymes; D012322:RNA Precursors; D012329:RNA, Bacterial; D043262:Ribonuclease P; D019793:Fluorescein