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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 of different pATSerUG derivatives with chemically synthesized variants of the P15 RNA at 160 mM Mg2+ as indicated. The experiments were performed under single-turnover conditions at pH 6.0 and at 37°C as described in ‘Materials and Methods’ section. The concentrations of substrates and P15 RNA variants were ≤20 nM and 39.1 µM, respectively. In cleavage of the +74 variants the reaction times were 4 h (lanes labeled 1), 24.5 h (lanes labeled 2) and 26 h (lanes labeled 3). C+74 (wild-type pATSerUG) and G+74 refer to the identity of the residue at position 74 in the substrate while C293 refers to the identity of the residue at position 293 (wild-type: G293) in the P15 RNA (for comparison we use Eco RPR numbering; Figure 1A). In the right panel pATSerUG was cleaved with chemically synthesized P15 RNA variants carrying substitutions at position 294 (Figure 1C). A reaction time of 18 h was used while assaying the different P15 variants. Controls (Ctrl) incubation of substrate in reaction buffer C without the P15 RNA; Ctrl I pATSerUG (26 h); Ctrl II pATSerUG(G+74) (26 h); Ctrl III pATSerUG (18 h).
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gkr1001-F5: Cleavage of different pATSerUG derivatives with chemically synthesized variants of the P15 RNA at 160 mM Mg2+ as indicated. The experiments were performed under single-turnover conditions at pH 6.0 and at 37°C as described in ‘Materials and Methods’ section. The concentrations of substrates and P15 RNA variants were ≤20 nM and 39.1 µM, respectively. In cleavage of the +74 variants the reaction times were 4 h (lanes labeled 1), 24.5 h (lanes labeled 2) and 26 h (lanes labeled 3). C+74 (wild-type pATSerUG) and G+74 refer to the identity of the residue at position 74 in the substrate while C293 refers to the identity of the residue at position 293 (wild-type: G293) in the P15 RNA (for comparison we use Eco RPR numbering; Figure 1A). In the right panel pATSerUG was cleaved with chemically synthesized P15 RNA variants carrying substitutions at position 294 (Figure 1C). A reaction time of 18 h was used while assaying the different P15 variants. Controls (Ctrl) incubation of substrate in reaction buffer C without the P15 RNA; Ctrl I pATSerUG (26 h); Ctrl II pATSerUG(G+74) (26 h); Ctrl III pATSerUG (18 h).

Mentions: The P15–P17 RNA and P15 RNA were generated using T7 DNA-dependent RNA polymerase. To eliminate the possibility that the RNA preparations contained trace amounts of Eco RPR, dot blot analysis and Reverse Transcription Polymerase Chain Reaction (RT-PCR) were performed (22). These two assays did not reveal any traces of Eco RPR in our small RNA preparations. Moreover, a chemically synthesized P15 RNA showed very similar cleavage properties as our original P15 RNA (not shown). In addition, substitution of residues participating in the RCCA–RNase P RNA, or RCCA–RPR, interaction (either in the P15 RNA or in the substrate and interacting residues underlined; Figure 2A and B) reduced cleavage compared to the ‘P15 RNA/wild-type pATSerUG’ situation (Figure 5; note that the P15 RNA variants were chemically synthesized). These results parallel our previous data using full-length Eco RPR and emphasizes the importance of the structural architecture of the RCCA–RPR interaction for catalysis (see also below; 24,29,37–41). From Figure 5 it is also apparent that the 2′-OH of U294 (Figure 2C) plays an important role for catalysis since replacement of this 2′-OH with a 2′-H resulted in a loss of activity under these conditions.Figure 5.


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

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

Cleavage of different pATSerUG derivatives with chemically synthesized variants of the P15 RNA at 160 mM Mg2+ as indicated. The experiments were performed under single-turnover conditions at pH 6.0 and at 37°C as described in ‘Materials and Methods’ section. The concentrations of substrates and P15 RNA variants were ≤20 nM and 39.1 µM, respectively. In cleavage of the +74 variants the reaction times were 4 h (lanes labeled 1), 24.5 h (lanes labeled 2) and 26 h (lanes labeled 3). C+74 (wild-type pATSerUG) and G+74 refer to the identity of the residue at position 74 in the substrate while C293 refers to the identity of the residue at position 293 (wild-type: G293) in the P15 RNA (for comparison we use Eco RPR numbering; Figure 1A). In the right panel pATSerUG was cleaved with chemically synthesized P15 RNA variants carrying substitutions at position 294 (Figure 1C). A reaction time of 18 h was used while assaying the different P15 variants. Controls (Ctrl) incubation of substrate in reaction buffer C without the P15 RNA; Ctrl I pATSerUG (26 h); Ctrl II pATSerUG(G+74) (26 h); Ctrl III pATSerUG (18 h).
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gkr1001-F5: Cleavage of different pATSerUG derivatives with chemically synthesized variants of the P15 RNA at 160 mM Mg2+ as indicated. The experiments were performed under single-turnover conditions at pH 6.0 and at 37°C as described in ‘Materials and Methods’ section. The concentrations of substrates and P15 RNA variants were ≤20 nM and 39.1 µM, respectively. In cleavage of the +74 variants the reaction times were 4 h (lanes labeled 1), 24.5 h (lanes labeled 2) and 26 h (lanes labeled 3). C+74 (wild-type pATSerUG) and G+74 refer to the identity of the residue at position 74 in the substrate while C293 refers to the identity of the residue at position 293 (wild-type: G293) in the P15 RNA (for comparison we use Eco RPR numbering; Figure 1A). In the right panel pATSerUG was cleaved with chemically synthesized P15 RNA variants carrying substitutions at position 294 (Figure 1C). A reaction time of 18 h was used while assaying the different P15 variants. Controls (Ctrl) incubation of substrate in reaction buffer C without the P15 RNA; Ctrl I pATSerUG (26 h); Ctrl II pATSerUG(G+74) (26 h); Ctrl III pATSerUG (18 h).
Mentions: The P15–P17 RNA and P15 RNA were generated using T7 DNA-dependent RNA polymerase. To eliminate the possibility that the RNA preparations contained trace amounts of Eco RPR, dot blot analysis and Reverse Transcription Polymerase Chain Reaction (RT-PCR) were performed (22). These two assays did not reveal any traces of Eco RPR in our small RNA preparations. Moreover, a chemically synthesized P15 RNA showed very similar cleavage properties as our original P15 RNA (not shown). In addition, substitution of residues participating in the RCCA–RNase P RNA, or RCCA–RPR, interaction (either in the P15 RNA or in the substrate and interacting residues underlined; Figure 2A and B) reduced cleavage compared to the ‘P15 RNA/wild-type pATSerUG’ situation (Figure 5; note that the P15 RNA variants were chemically synthesized). These results parallel our previous data using full-length Eco RPR and emphasizes the importance of the structural architecture of the RCCA–RPR interaction for catalysis (see also below; 24,29,37–41). From Figure 5 it is also apparent that the 2′-OH of U294 (Figure 2C) plays an important role for catalysis since replacement of this 2′-OH with a 2′-H resulted in a loss of activity under these conditions.Figure 5.

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