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Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site.

Kikovska E, Brännvall M, Kufel J, Kirsebom LA - Nucleic Acids Res. (2005)

Bottom Line: These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step.In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA.Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.

View Article: PubMed Central - PubMed

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

ABSTRACT
Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5' termini by cleaving tRNA precursors immediately 5' of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.

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Illustration of the ‘RCCA–RNase P RNA interaction’ (interacting residues underlined) and the interaction between A248 and residue −1 in the substrate, the ‘A248/N−1 interaction’ [for details see the text and (14,22), and references therein]. The residues indicated in red correspond to those that were replaced in this report. The residues in red circles show the ‘+73/294 interaction’ while the arrow mark the canonical RNase P cleavage site at +1. In the case of the C−1 variants, C−1 is inferred to base pair with G+73 in the absence of RNase P RNA. A and B (in gray circles) represent Mg2+ ions that have been suggested to be positioned at and in the vicinity of the cleavage site [(22) and references therein] while C is an additional ion positioned in the P15-loop. The 2′-OH at the −1 position has been suggested coordinating Mg2+ at the cleavage site, and/or interacting with RNase P RNA (14,15,22,28,29,31,34).
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fig5: Illustration of the ‘RCCA–RNase P RNA interaction’ (interacting residues underlined) and the interaction between A248 and residue −1 in the substrate, the ‘A248/N−1 interaction’ [for details see the text and (14,22), and references therein]. The residues indicated in red correspond to those that were replaced in this report. The residues in red circles show the ‘+73/294 interaction’ while the arrow mark the canonical RNase P cleavage site at +1. In the case of the C−1 variants, C−1 is inferred to base pair with G+73 in the absence of RNase P RNA. A and B (in gray circles) represent Mg2+ ions that have been suggested to be positioned at and in the vicinity of the cleavage site [(22) and references therein] while C is an additional ion positioned in the P15-loop. The 2′-OH at the −1 position has been suggested coordinating Mg2+ at the cleavage site, and/or interacting with RNase P RNA (14,15,22,28,29,31,34).

Mentions: The C−1 derivatives pATSerCGG/C and pATSerCGU/A were cleaved with lower rates relative to the corresponding U−1 derivatives irrespective of [Mg2+] indicating the importance of the −1 residue (and/or +73, see below). Comparing kobs for cleavage of the U+1DAP+72 variants suggested an increased rate of cleavage with a C at position −1. Moreover, in contrast to what we observed for the U−1-variants, no rescue in the rate of cleavage was observed for the C−1 derivatives as a result of increasing [Mg2+] under the conditions tested. It is conceivable that this difference is influenced by the fact that C−1 is base-paired to G+73 in the substrate (see Figure 5).


Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site.

Kikovska E, Brännvall M, Kufel J, Kirsebom LA - Nucleic Acids Res. (2005)

Illustration of the ‘RCCA–RNase P RNA interaction’ (interacting residues underlined) and the interaction between A248 and residue −1 in the substrate, the ‘A248/N−1 interaction’ [for details see the text and (14,22), and references therein]. The residues indicated in red correspond to those that were replaced in this report. The residues in red circles show the ‘+73/294 interaction’ while the arrow mark the canonical RNase P cleavage site at +1. In the case of the C−1 variants, C−1 is inferred to base pair with G+73 in the absence of RNase P RNA. A and B (in gray circles) represent Mg2+ ions that have been suggested to be positioned at and in the vicinity of the cleavage site [(22) and references therein] while C is an additional ion positioned in the P15-loop. The 2′-OH at the −1 position has been suggested coordinating Mg2+ at the cleavage site, and/or interacting with RNase P RNA (14,15,22,28,29,31,34).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074746&req=5

fig5: Illustration of the ‘RCCA–RNase P RNA interaction’ (interacting residues underlined) and the interaction between A248 and residue −1 in the substrate, the ‘A248/N−1 interaction’ [for details see the text and (14,22), and references therein]. The residues indicated in red correspond to those that were replaced in this report. The residues in red circles show the ‘+73/294 interaction’ while the arrow mark the canonical RNase P cleavage site at +1. In the case of the C−1 variants, C−1 is inferred to base pair with G+73 in the absence of RNase P RNA. A and B (in gray circles) represent Mg2+ ions that have been suggested to be positioned at and in the vicinity of the cleavage site [(22) and references therein] while C is an additional ion positioned in the P15-loop. The 2′-OH at the −1 position has been suggested coordinating Mg2+ at the cleavage site, and/or interacting with RNase P RNA (14,15,22,28,29,31,34).
Mentions: The C−1 derivatives pATSerCGG/C and pATSerCGU/A were cleaved with lower rates relative to the corresponding U−1 derivatives irrespective of [Mg2+] indicating the importance of the −1 residue (and/or +73, see below). Comparing kobs for cleavage of the U+1DAP+72 variants suggested an increased rate of cleavage with a C at position −1. Moreover, in contrast to what we observed for the U−1-variants, no rescue in the rate of cleavage was observed for the C−1 derivatives as a result of increasing [Mg2+] under the conditions tested. It is conceivable that this difference is influenced by the fact that C−1 is base-paired to G+73 in the substrate (see Figure 5).

Bottom Line: These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step.In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA.Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.

View Article: PubMed Central - PubMed

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

ABSTRACT
Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5' termini by cleaving tRNA precursors immediately 5' of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.

Show MeSH
Related in: MedlinePlus