Structural perspective on the activation of RNAse P RNA by protein.

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Nature Structural & Molecular Biology (Impact Factor: 11.63). 12/2005; 12(11):958-64. DOI: 10.1038/nsmb1004
Source: PubMed

ABSTRACT Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5' maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.

  • Source
    Bulletin of The Korean Chemical Society. 01/2010; 31(7):2081-2084.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNase P is an RNA-based enzyme primarily responsible for 5'-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNA(Phe) revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52-57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the 'RNR' region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N(-4) and N(-5) nucleotides of the pre-tRNA 5'-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.
    Nucleic Acids Research 08/2012; · 8.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A single enzyme, ribonuclease P (RNase P), processes the 5 prime ends of tRNA precursors (ptRNA) in cells and organelles that carry out tRNA biosynthesis. This substrate population includes over 80 different competing ptRNAs in Escherichia coli. While the reaction kinetics and molecular recognition of a few individual model substrates of bacterial RNase P have been well described, the competitive substrate kinetics of the enzyme are comparatively unexplored. To understand the factors that determine how different ptRNA substrates compete for processing by E. coli RNase P, we compared the steady state reaction kinetics of two ptRNAs that differ at sequences that are contacted by the enzyme. For both ptRNAs, substrate cleavage is fast relative to dissociation. As a consequence, V/K, the rate constant for the reaction at limiting substrate concentrations, reflects the substrate association step for both ptRNAs. Reactions containing two or more ptRNAs follow simple competitive alternative substrate kinetics in which the relative rates of processing are determined by ptRNA concentration and their V/K. The relative V/K values for eight different ptRNAs, that were selected to represent the range of structure variation at sites contacted by RNase P, were determined by internal competition in reactions in which all eight substrates were present simultaneously. The results reveal a relatively narrow range of V/K values suggesting that rates of ptRNA processing by RNase P are tuned for uniform specificity and consequently optimal coupling to precursor biosynthesis.
    Journal of Biological Chemistry 01/2013; · 4.60 Impact Factor


1 Download
Available from