The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit.
The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence. Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P x pre-tRNA(Asp) complex. The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNA(Asp). Nor does the protein component alter the pH dependence of pre-tRNA(Asp) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state. However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme x pre-tRNA complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P x pre-tRNA complex indirectly by binding and positioning pre-tRNA. Furthermore, RNase P is inhibited by cobalt hexammine (K(I) = 0.11 +/- 0.01 mM) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P. These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.
Kurz
Jeffrey C
JC
Department of Chemistry, University of Michigan, 930 North University, Ann Arbor, Michigan 48109-1055, USA.
Fierke
Carol A
CA
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D003035:Cobalt / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012349:RNA, Transfer, Asp / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*
D012322:RNA Precursors; D012349:RNA, Transfer, Asp; C032300:hexaamminecobalt(II); D003035:Cobalt; D012260:Ribonucleases; D008274:Magnesium