title: The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer.
abstract: Ribonuclease P (RNase P) catalyzes the metal-dependent 5' end maturation of precursor tRNAs (pre-tRNAs). In Bacteria, RNase P is composed of a catalytic RNA (PRNA) and a protein subunit (P protein) necessary for function in vivo. The P protein enhances pre-tRNA affinity, selectivity, and cleavage efficiency, as well as modulates the cation requirement for RNase P function. Bacterial P proteins share little sequence conservation although the protein structures are homologous. Here we combine site-directed mutagenesis, affinity measurements, and single turnover kinetics to demonstrate that two residues (R60 and R62) in the most highly conserved region of the P protein, the RNR motif (R60-R68 in Bacillus subtilis), stabilize PRNA complexes with both P protein (PRNA•P protein) and pre-tRNA (PRNA•P protein•pre-tRNA). Additionally, these data indicate that the RNR motif enhances a metal-stabilized conformational change in RNase P that accompanies substrate binding and is essential for efficient catalysis. Stabilization of this conformational change contributes to both the decreased metal requirement and the enhanced substrate recognition of the RNase P holoenzyme, illuminating the role of the most highly conserved region of P protein in the RNase P reaction pathway.
authors.affiliation: Chemistry Department, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Kristin S
authors.initials: KS
authors.lastname: Koutmou
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Day-Storms
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation / Q000502:physiology; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D020871:RNA Stability / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013379:Substrate Specificity
chemical_list: D046911:Macromolecular Substances; D012322:RNA Precursors; D012313:RNA; D043262:Ribonuclease P