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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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(A) Cleavage of pATSerUG with wild-type Eco RPR and P15 RNA expressed as a percentage of cleavage per min as a function of Mg2+. Concentration of: substrate ≤20 nM, wild-type Eco RPR 3.2 µM and P15 RNA 39.1 µM. Data are the average of two independent experiments. Bars indicate the experimental range. (B) Cleavage of pATSerUG in percentage as a function of time and accumulation of the 5′-cleavage fragment over time as indicated. Same concentrations of P15 RNA and substrate as in Figure 3 were used. (C) A typical experiment illustrating cleavage of pATSerUG with P15 RNA as a function of increasing concentration of P15 RNA. The concentration of substrate was ≤20 nM and the reaction time after mixing of substrate and P15 RNA was 25 h.
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gkr1001-F4: (A) Cleavage of pATSerUG with wild-type Eco RPR and P15 RNA expressed as a percentage of cleavage per min as a function of Mg2+. Concentration of: substrate ≤20 nM, wild-type Eco RPR 3.2 µM and P15 RNA 39.1 µM. Data are the average of two independent experiments. Bars indicate the experimental range. (B) Cleavage of pATSerUG in percentage as a function of time and accumulation of the 5′-cleavage fragment over time as indicated. Same concentrations of P15 RNA and substrate as in Figure 3 were used. (C) A typical experiment illustrating cleavage of pATSerUG with P15 RNA as a function of increasing concentration of P15 RNA. The concentration of substrate was ≤20 nM and the reaction time after mixing of substrate and P15 RNA was 25 h.

Mentions: Comparing the Mg2+ profiles in cleavage of pATSerUG revealed that wild-type Eco RPR and the P15 RNA have similar Mg2+ requirements with a plateau around 160 mM. However, at higher [Mg2+], the rate of cleavage for the P15 RNA was lower compared to cleavage at 160 mM (Figure 4A). Moreover, the percentage of cleavage of pATSerUG with P15 RNA increased linearly over time (Figure 4B) and with increasing concentration of ribozyme (Figure 4C; complete cleavage in any of our catalytic RNA substrate combinations was not observed and maximum fraction of substrate converted into product was ~10%). From the data shown in Figure 2C, it is evident that the rate does not plateau at the highest concentration of P15 RNA tested. Therefore, the data could not be fit to obtain the kobs value. The absence of saturable behavior might be due to substrate binding being rate limiting and/or P15 RNA folding being sub-optimal. We know from our earlier NMR spectroscopy studies that the P15 RNA folds into different structures in solution however we do not know which of the structures are catalytically active (35). Although, the kobs value for H1 RNA is 2.6 × 10−6 min−1 (22), i.e. 106–107-fold lower compared to Eco RPR (Supplementary Table S2). On the basis of that observation, the percentage of cleavage of pATSerUG by P15 RNA is similar to that observed for H1 RNA under these conditions; therefore, we estimate that the rate of cleavage for P15 RNA is roughly in the same range as for H1 RNA. Moreover, we also note that the P15–P17 RNA appears to be more active than the P15 RNA and this might be due to the increased flexibility of the P15 RNA structure. It is also conceivable, but not mutually exclusive, that the larger P15–P17 RNA interacts more productively with the 5′-leader of the substrate.Figure 4.


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

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

(A) Cleavage of pATSerUG with wild-type Eco RPR and P15 RNA expressed as a percentage of cleavage per min as a function of Mg2+. Concentration of: substrate ≤20 nM, wild-type Eco RPR 3.2 µM and P15 RNA 39.1 µM. Data are the average of two independent experiments. Bars indicate the experimental range. (B) Cleavage of pATSerUG in percentage as a function of time and accumulation of the 5′-cleavage fragment over time as indicated. Same concentrations of P15 RNA and substrate as in Figure 3 were used. (C) A typical experiment illustrating cleavage of pATSerUG with P15 RNA as a function of increasing concentration of P15 RNA. The concentration of substrate was ≤20 nM and the reaction time after mixing of substrate and P15 RNA was 25 h.
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Related In: Results  -  Collection

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gkr1001-F4: (A) Cleavage of pATSerUG with wild-type Eco RPR and P15 RNA expressed as a percentage of cleavage per min as a function of Mg2+. Concentration of: substrate ≤20 nM, wild-type Eco RPR 3.2 µM and P15 RNA 39.1 µM. Data are the average of two independent experiments. Bars indicate the experimental range. (B) Cleavage of pATSerUG in percentage as a function of time and accumulation of the 5′-cleavage fragment over time as indicated. Same concentrations of P15 RNA and substrate as in Figure 3 were used. (C) A typical experiment illustrating cleavage of pATSerUG with P15 RNA as a function of increasing concentration of P15 RNA. The concentration of substrate was ≤20 nM and the reaction time after mixing of substrate and P15 RNA was 25 h.
Mentions: Comparing the Mg2+ profiles in cleavage of pATSerUG revealed that wild-type Eco RPR and the P15 RNA have similar Mg2+ requirements with a plateau around 160 mM. However, at higher [Mg2+], the rate of cleavage for the P15 RNA was lower compared to cleavage at 160 mM (Figure 4A). Moreover, the percentage of cleavage of pATSerUG with P15 RNA increased linearly over time (Figure 4B) and with increasing concentration of ribozyme (Figure 4C; complete cleavage in any of our catalytic RNA substrate combinations was not observed and maximum fraction of substrate converted into product was ~10%). From the data shown in Figure 2C, it is evident that the rate does not plateau at the highest concentration of P15 RNA tested. Therefore, the data could not be fit to obtain the kobs value. The absence of saturable behavior might be due to substrate binding being rate limiting and/or P15 RNA folding being sub-optimal. We know from our earlier NMR spectroscopy studies that the P15 RNA folds into different structures in solution however we do not know which of the structures are catalytically active (35). Although, the kobs value for H1 RNA is 2.6 × 10−6 min−1 (22), i.e. 106–107-fold lower compared to Eco RPR (Supplementary Table S2). On the basis of that observation, the percentage of cleavage of pATSerUG by P15 RNA is similar to that observed for H1 RNA under these conditions; therefore, we estimate that the rate of cleavage for P15 RNA is roughly in the same range as for H1 RNA. Moreover, we also note that the P15–P17 RNA appears to be more active than the P15 RNA and this might be due to the increased flexibility of the P15 RNA structure. It is also conceivable, but not mutually exclusive, that the larger P15–P17 RNA interacts more productively with the 5′-leader of the substrate.Figure 4.

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