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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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Cleavage activities of pATSerUG for the Eco RPR C domain with and without the P15–P17 domain (left panel), and for wt Eco RPR and Eco RPRΔP15–P17 (right panel). Amount of RNA added: C construct 7 µM and CP construct 5.5 µM. For wt Eco RPR and Eco RPRΔP15–P17, we used 0.8 µM and 2.8 µM, respectively. Irrespective of RPR construct the concentration of γ-32P 5′-end labeled pATSerUG was ≤20 nM. Time of incubations were: 4 h for the Eco RPR C-domain variants, 0.5 min for wt Eco RPR and 4 h for Eco RPRΔP15–P17. Ctrl (control): incubation of pATSerUG alone for 4 h in the reaction buffer C.
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gkr1001-F6: Cleavage activities of pATSerUG for the Eco RPR C domain with and without the P15–P17 domain (left panel), and for wt Eco RPR and Eco RPRΔP15–P17 (right panel). Amount of RNA added: C construct 7 µM and CP construct 5.5 µM. For wt Eco RPR and Eco RPRΔP15–P17, we used 0.8 µM and 2.8 µM, respectively. Irrespective of RPR construct the concentration of γ-32P 5′-end labeled pATSerUG was ≤20 nM. Time of incubations were: 4 h for the Eco RPR C-domain variants, 0.5 min for wt Eco RPR and 4 h for Eco RPRΔP15–P17. Ctrl (control): incubation of pATSerUG alone for 4 h in the reaction buffer C.

Mentions: RNase P RNA consists of the catalytic (C) and the specificity (S) domains [Figure 1 (15,16)] where the C domain alone mediates cleavage in the absence and in the presence of the RNase P protein with significantly reduced activity (44,45). To investigate the role of the P15–P17 domain in relationship with the C domain, we tested constructs with and without the P15–P17 domain (Figure 1 and Supplementary Figure S1). Figure 6 shows that the C domain lacking P15–P17 (C construct) did not promote any detectable cleavage of pATSerUG under these conditions at 160 mM Mg2+ while its presence did (CP construct). The kobs for the CP construct was similar to the kobs previously determined for Eco RPRΔP15–P17 RNA (Supplementary Table S2) and in keeping with rates determined for cleavage using a C domain derived from a type B RPR (45). Note also that deleting P15–P17 in Eco RPR result in a catalyst with significantly reduced activity (Figure 6) in keeping with our previous findings (22; Supplementary Table S2). These data show that the C domain of type A mediates cleavage in the absence of protein and further corroborate the importance of the P15–P17 domain and its role for efficient catalysis. In this context, we note that the S domain is almost completely missing in some archaeal RPRs and these RPRs do mediate cleavage in the absence of proteins (46).Figure 6.


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

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

Cleavage activities of pATSerUG for the Eco RPR C domain with and without the P15–P17 domain (left panel), and for wt Eco RPR and Eco RPRΔP15–P17 (right panel). Amount of RNA added: C construct 7 µM and CP construct 5.5 µM. For wt Eco RPR and Eco RPRΔP15–P17, we used 0.8 µM and 2.8 µM, respectively. Irrespective of RPR construct the concentration of γ-32P 5′-end labeled pATSerUG was ≤20 nM. Time of incubations were: 4 h for the Eco RPR C-domain variants, 0.5 min for wt Eco RPR and 4 h for Eco RPRΔP15–P17. Ctrl (control): incubation of pATSerUG alone for 4 h in the reaction buffer C.
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gkr1001-F6: Cleavage activities of pATSerUG for the Eco RPR C domain with and without the P15–P17 domain (left panel), and for wt Eco RPR and Eco RPRΔP15–P17 (right panel). Amount of RNA added: C construct 7 µM and CP construct 5.5 µM. For wt Eco RPR and Eco RPRΔP15–P17, we used 0.8 µM and 2.8 µM, respectively. Irrespective of RPR construct the concentration of γ-32P 5′-end labeled pATSerUG was ≤20 nM. Time of incubations were: 4 h for the Eco RPR C-domain variants, 0.5 min for wt Eco RPR and 4 h for Eco RPRΔP15–P17. Ctrl (control): incubation of pATSerUG alone for 4 h in the reaction buffer C.
Mentions: RNase P RNA consists of the catalytic (C) and the specificity (S) domains [Figure 1 (15,16)] where the C domain alone mediates cleavage in the absence and in the presence of the RNase P protein with significantly reduced activity (44,45). To investigate the role of the P15–P17 domain in relationship with the C domain, we tested constructs with and without the P15–P17 domain (Figure 1 and Supplementary Figure S1). Figure 6 shows that the C domain lacking P15–P17 (C construct) did not promote any detectable cleavage of pATSerUG under these conditions at 160 mM Mg2+ while its presence did (CP construct). The kobs for the CP construct was similar to the kobs previously determined for Eco RPRΔP15–P17 RNA (Supplementary Table S2) and in keeping with rates determined for cleavage using a C domain derived from a type B RPR (45). Note also that deleting P15–P17 in Eco RPR result in a catalyst with significantly reduced activity (Figure 6) in keeping with our previous findings (22; Supplementary Table S2). These data show that the C domain of type A mediates cleavage in the absence of protein and further corroborate the importance of the P15–P17 domain and its role for efficient catalysis. In this context, we note that the S domain is almost completely missing in some archaeal RPRs and these RPRs do mediate cleavage in the absence of proteins (46).Figure 6.

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