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: 13.31). 12/2005; 12(11):958-64. DOI: 10.1038/nsmb1004
Source: PubMed


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.

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Available from: Amy H Buck, Apr 14, 2015
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    • "By comparison to group I ribozyme structures, the RNase P structure sheds light on the characteristics of a true RNA enzyme in terms of substrate binding, discrimination between the substrate and the product—by a protein ''cofactor,'' and organization of the catalytic site. The structure supports a view of the holoenzyme that is finally very close to what was deduced qualitatively from the accumulated biochemical and theoretical data that led to molecular models published >5 yr ago (Tsai et al. 2003; Buck et al. 2005). Yet, the fairly low resolution of the structure together with the fact that the tRNA product is actually bound to the holoenzyme instead of the pre-tRNA substrate still leaves open some questions related to the precise molecular interactions taking place between the components of the system and related to the mechanism and the dynamics of the catalysis. "
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    ABSTRACT: Apart from the ribosome, the crystal structure of the bacterial RNase P in complex with a tRNA, reported by Reiter and colleagues recently, constitutes the first example of a multiple turnover RNA enzyme. Except in rare exceptions, RNase P is ubiquitous and, like the ribosome, is older than the initial branch point of the phylogenetic tree. Importantly, the structure shows how the RNA and the protein moieties cooperate to process the pre-tRNA substrates. The catalytic site comprises some critical RNA residues spread over the secondary structure but gathered in a compact volume next to the protein, which helps recognize and orient the substrate. The discussion here outlines some important aspects of that crystal structure, some of which could apply to RNA molecules in general.
    RNA 09/2011; 17(9):1615-8. DOI:10.1261/rna.2841511 · 4.94 Impact Factor
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    • "Previous melting, binding, and folding studies of E. coli and B. subtilis RNase P suggest that bacterial proteins stabilize local PRNA structure (Buck et al. 2005a). Footprinting studies of the E. coli and B. stearothermophilus PRNAs complexed with E. coli, B. stearothermophilus, or B. subtilis protein revealed that all three bacterial proteins protect the same PRNA region, including nucleotides in helices P3 and P4 (Buck et al. 2005b). Consistent with this, affinity cleavage data indicate that the RNR motif is in close proximity to PRNA helices P2, P3, P4, and P19 (Niranjanakumari et al. 2007). "
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    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.
    RNA 07/2011; 17(7):1225-35. DOI:10.1261/rna.2742511 · 4.94 Impact Factor
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    • "The occurrence of multiple conformers of the Atu RNA indicates, as seen for other RNAs, that much of the molecular population is kinetically trapped, preventing proper folding (Pan and Sosnick 1997; Pan et al. 1999; Buck et al. 2005b). Since ionic strength influences folding of some RNase P RNAs and high ionic strength is required for catalytic activity of RNase P RNA (Guerrier-Takada et al. 1983; Siegel et al. "
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    ABSTRACT: The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.
    RNA 06/2011; 17(6):1159-71. DOI:10.1261/rna.2563511 · 4.94 Impact Factor
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