The Affinity of Magnesium Binding Sites in the Bacillus subtilis Rnase PPre-tRna Complex Is Enhanced by the Protein Subunit

Kurz, J.C.; Fierke, C.A.

Biochemistry 41(30): 9545-9558


ISSN/ISBN: 0006-2960
DOI: 10.1021/bi025553w
Accession: 067188587

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The Rna subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of t Rna 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor t Rna in vivo, enhances pre-t Rna 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·pre-t RNAAsp 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 t RNAAsp. Nor does the protein component alter the p H dependence of pre-t RNAAsp 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-t Rna binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme·pre-t Rna complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P·pre-t Rna complex indirectly by binding and positioning pre-t RNA. Furthermore, RNase P is inhibited by cobalt hexammine (Ki = 0.11 ± 0.01 m M) 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.