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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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Cleavage of pATSerCGG/C and pATSerCGU/A by M1 RNA at different concentrations of Mg2+ (A) and [Mg2+]/([Mg2+] + [Mn2+]) ratios (B). (A) Cleavage rates pATSerCGG/C and pATSerCGU/A expressed as percentage of cleavage per minute. (B) Normalized miscleavage at −1 of pATSerCGG/C and pATSerCGU/A as a function of [Mg2+]/([Mg2+] + [Mn2+]) as indicated. The total divalent metal ion concentration was kept constant at 40 mM and the concentrations of Mg2+ and Mn2+ were varied as indicated. The curves are averages of several independent experiments and the bars indicate experimental errors. The experiments were performed at 37°C as outlined in Materials and Methods under single turnover conditions at pH 6.1. pATSerCGG/C = triangles; pATSerCGU/A = squares.
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fig4: Cleavage of pATSerCGG/C and pATSerCGU/A by M1 RNA at different concentrations of Mg2+ (A) and [Mg2+]/([Mg2+] + [Mn2+]) ratios (B). (A) Cleavage rates pATSerCGG/C and pATSerCGU/A expressed as percentage of cleavage per minute. (B) Normalized miscleavage at −1 of pATSerCGG/C and pATSerCGU/A as a function of [Mg2+]/([Mg2+] + [Mn2+]) as indicated. The total divalent metal ion concentration was kept constant at 40 mM and the concentrations of Mg2+ and Mn2+ were varied as indicated. The curves are averages of several independent experiments and the bars indicate experimental errors. The experiments were performed at 37°C as outlined in Materials and Methods under single turnover conditions at pH 6.1. pATSerCGG/C = triangles; pATSerCGU/A = squares.

Mentions: At pH 7.2, chemistry is not rate limiting, therefore these data do not provide information about the importance of the G+1C+72 to U+1A+72 change with respect to the chemistry of cleavage. We therefore determined the rate constant kobs for cleavage under single turnover conditions at pH 6.1 where chemistry of cleavage is suggested to be rate limiting [(22,31–34); see also Table 2 legend]. Furthermore, our initial experiments where we studied rate of cleavage under single turnover conditions as a function of increasing Mg2+-concentration indicated different Mg2+ requirements due to replacing G+1C+72 with U+1A+72 (Figure 4A). We therefore determined kobs at different Mg2+ concentrations. The results are summarized in Table 2.


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)

Cleavage of pATSerCGG/C and pATSerCGU/A by M1 RNA at different concentrations of Mg2+ (A) and [Mg2+]/([Mg2+] + [Mn2+]) ratios (B). (A) Cleavage rates pATSerCGG/C and pATSerCGU/A expressed as percentage of cleavage per minute. (B) Normalized miscleavage at −1 of pATSerCGG/C and pATSerCGU/A as a function of [Mg2+]/([Mg2+] + [Mn2+]) as indicated. The total divalent metal ion concentration was kept constant at 40 mM and the concentrations of Mg2+ and Mn2+ were varied as indicated. The curves are averages of several independent experiments and the bars indicate experimental errors. The experiments were performed at 37°C as outlined in Materials and Methods under single turnover conditions at pH 6.1. pATSerCGG/C = triangles; pATSerCGU/A = squares.
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Related In: Results  -  Collection

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fig4: Cleavage of pATSerCGG/C and pATSerCGU/A by M1 RNA at different concentrations of Mg2+ (A) and [Mg2+]/([Mg2+] + [Mn2+]) ratios (B). (A) Cleavage rates pATSerCGG/C and pATSerCGU/A expressed as percentage of cleavage per minute. (B) Normalized miscleavage at −1 of pATSerCGG/C and pATSerCGU/A as a function of [Mg2+]/([Mg2+] + [Mn2+]) as indicated. The total divalent metal ion concentration was kept constant at 40 mM and the concentrations of Mg2+ and Mn2+ were varied as indicated. The curves are averages of several independent experiments and the bars indicate experimental errors. The experiments were performed at 37°C as outlined in Materials and Methods under single turnover conditions at pH 6.1. pATSerCGG/C = triangles; pATSerCGU/A = squares.
Mentions: At pH 7.2, chemistry is not rate limiting, therefore these data do not provide information about the importance of the G+1C+72 to U+1A+72 change with respect to the chemistry of cleavage. We therefore determined the rate constant kobs for cleavage under single turnover conditions at pH 6.1 where chemistry of cleavage is suggested to be rate limiting [(22,31–34); see also Table 2 legend]. Furthermore, our initial experiments where we studied rate of cleavage under single turnover conditions as a function of increasing Mg2+-concentration indicated different Mg2+ requirements due to replacing G+1C+72 with U+1A+72 (Figure 4A). We therefore determined kobs at different Mg2+ concentrations. The results are summarized in Table 2.

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