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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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Kinetic constants of various pATSerCG derivatives cleaved by M1 RNA under multiple turnover conditions as indicated. The constants were determined at pH 7.2 as described in Materials and Methods. Given kcat and Km values are averages of several independent experiments and experimental errors are given as ‘±’. The kcat/Km values were calculated using the kcat and Km numbers.
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fig3: Kinetic constants of various pATSerCG derivatives cleaved by M1 RNA under multiple turnover conditions as indicated. The constants were determined at pH 7.2 as described in Materials and Methods. Given kcat and Km values are averages of several independent experiments and experimental errors are given as ‘±’. The kcat/Km values were calculated using the kcat and Km numbers.

Mentions: We previously reported the kinetic constants for cleavage of several pATSerCG variants under multiple turnover conditions at pH 7.2 (12,13,23,27). Hence, we first determined cleavage efficiency for the various +1/+72 derivatives by calculating kcat/Km values based on determination of kcat and Km under multiple turnover conditions at pH 7.2 and 160 mM Mg2+ (see Materials and Methods). As shown in Figure 3, reductions ranging between 16- and ∼150-fold in kcat/Km (for cleavage at +1) for pATSerCGU/DAP and pATSerCGU/A, respectively, were observed compared to cleavage of the corresponding G+1C+72 variant. This was mainly due to an increased Km (≈10-fold) for the former, while for pATSerCGU/A kcat was down 5-fold and Km was increased 30-fold. In keeping with our previous data (13), pATSerUGG/C was cleaved with a higher efficiency compared to cleavage of pATSerCGG/C indicating the importance of the identity of residue −1 (Figure 3). These data indicated that replacement of G+1C+72 with U+1A+72 (or U+1DAP+72) influence the efficiency of cleavage also in the context of the model substrate, pATSer.


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)

Kinetic constants of various pATSerCG derivatives cleaved by M1 RNA under multiple turnover conditions as indicated. The constants were determined at pH 7.2 as described in Materials and Methods. Given kcat and Km values are averages of several independent experiments and experimental errors are given as ‘±’. The kcat/Km values were calculated using the kcat and Km numbers.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Kinetic constants of various pATSerCG derivatives cleaved by M1 RNA under multiple turnover conditions as indicated. The constants were determined at pH 7.2 as described in Materials and Methods. Given kcat and Km values are averages of several independent experiments and experimental errors are given as ‘±’. The kcat/Km values were calculated using the kcat and Km numbers.
Mentions: We previously reported the kinetic constants for cleavage of several pATSerCG variants under multiple turnover conditions at pH 7.2 (12,13,23,27). Hence, we first determined cleavage efficiency for the various +1/+72 derivatives by calculating kcat/Km values based on determination of kcat and Km under multiple turnover conditions at pH 7.2 and 160 mM Mg2+ (see Materials and Methods). As shown in Figure 3, reductions ranging between 16- and ∼150-fold in kcat/Km (for cleavage at +1) for pATSerCGU/DAP and pATSerCGU/A, respectively, were observed compared to cleavage of the corresponding G+1C+72 variant. This was mainly due to an increased Km (≈10-fold) for the former, while for pATSerCGU/A kcat was down 5-fold and Km was increased 30-fold. In keeping with our previous data (13), pATSerUGG/C was cleaved with a higher efficiency compared to cleavage of pATSerCGG/C indicating the importance of the identity of residue −1 (Figure 3). These data indicated that replacement of G+1C+72 with U+1A+72 (or U+1DAP+72) influence the efficiency of cleavage also in the context of the model substrate, pATSer.

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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