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Cleavage mediated by the P15 domain of bacterial RNase P RNA.

Kikovska E, Wu S, Mao G, Kirsebom LA - Nucleic Acids Res. (2011)

Bottom Line: One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex.The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg(2+) at the cleavage site.Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden.

ABSTRACT
Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg(2+) at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for 'RNA-mediated' cleavage of RNA.

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Predicted secondary structures of wild-type Eco RPR and human RPR, H1 RNA (58). The specificity (S) and catalytic (C) domains are separated with the dashed line and the Eco RPR P15–P17 domain is highlighted in light grey. The Eco RPR P15–P17 domain was introduced into H1 RNA generating H1 RNA + Eco P15–P17 as indicated at the position marked with a grey box. Eco RPRΔP15–P17 carries a deletion of P15–P17 except the GU pair marked with black circles (22). The highlighted regions in dark grey mark regions in RPR (including P15–P17) known to be important for binding of the substrate and that show structural differences, for details see main text. P refers to helices in Eco RPR and H1 RNA.
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gkr1001-F1: Predicted secondary structures of wild-type Eco RPR and human RPR, H1 RNA (58). The specificity (S) and catalytic (C) domains are separated with the dashed line and the Eco RPR P15–P17 domain is highlighted in light grey. The Eco RPR P15–P17 domain was introduced into H1 RNA generating H1 RNA + Eco P15–P17 as indicated at the position marked with a grey box. Eco RPRΔP15–P17 carries a deletion of P15–P17 except the GU pair marked with black circles (22). The highlighted regions in dark grey mark regions in RPR (including P15–P17) known to be important for binding of the substrate and that show structural differences, for details see main text. P refers to helices in Eco RPR and H1 RNA.

Mentions: Like proteins, RNAs are composed of different domains and these can have different functions, for example one domain can bind a small ligand or cofactor while another constitutes the active site (1; see also Refs 2 and 3). When separated, the domains can fold in a similar way compared to the fold they have in the full-length RNA molecule. This is exemplified by the P4-P6 domain of the Tetrahymena group I intron (4–6; for a review see, e.g. Ref. 7) and the group II intron where domain five retains its catalytic activity when separated from the full-length RNA (8–12; for a review see, e.g. Ref. 13). This is also true for the catalytic RNA component (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors [Figure 1 (14–21)].Figure 1.


Cleavage mediated by the P15 domain of bacterial RNase P RNA.

Kikovska E, Wu S, Mao G, Kirsebom LA - Nucleic Acids Res. (2011)

Predicted secondary structures of wild-type Eco RPR and human RPR, H1 RNA (58). The specificity (S) and catalytic (C) domains are separated with the dashed line and the Eco RPR P15–P17 domain is highlighted in light grey. The Eco RPR P15–P17 domain was introduced into H1 RNA generating H1 RNA + Eco P15–P17 as indicated at the position marked with a grey box. Eco RPRΔP15–P17 carries a deletion of P15–P17 except the GU pair marked with black circles (22). The highlighted regions in dark grey mark regions in RPR (including P15–P17) known to be important for binding of the substrate and that show structural differences, for details see main text. P refers to helices in Eco RPR and H1 RNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299987&req=5

gkr1001-F1: Predicted secondary structures of wild-type Eco RPR and human RPR, H1 RNA (58). The specificity (S) and catalytic (C) domains are separated with the dashed line and the Eco RPR P15–P17 domain is highlighted in light grey. The Eco RPR P15–P17 domain was introduced into H1 RNA generating H1 RNA + Eco P15–P17 as indicated at the position marked with a grey box. Eco RPRΔP15–P17 carries a deletion of P15–P17 except the GU pair marked with black circles (22). The highlighted regions in dark grey mark regions in RPR (including P15–P17) known to be important for binding of the substrate and that show structural differences, for details see main text. P refers to helices in Eco RPR and H1 RNA.
Mentions: Like proteins, RNAs are composed of different domains and these can have different functions, for example one domain can bind a small ligand or cofactor while another constitutes the active site (1; see also Refs 2 and 3). When separated, the domains can fold in a similar way compared to the fold they have in the full-length RNA molecule. This is exemplified by the P4-P6 domain of the Tetrahymena group I intron (4–6; for a review see, e.g. Ref. 7) and the group II intron where domain five retains its catalytic activity when separated from the full-length RNA (8–12; for a review see, e.g. Ref. 13). This is also true for the catalytic RNA component (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors [Figure 1 (14–21)].Figure 1.

Bottom Line: One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex.The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg(2+) at the cleavage site.Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden.

ABSTRACT
Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg(2+) at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for 'RNA-mediated' cleavage of RNA.

Show MeSH