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Deficiency of the tRNATyr:Psi 35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery.

Muller S, Urban A, Hecker A, Leclerc F, Branlant C, Motorin Y - Nucleic Acids Res. (2009)

Bottom Line: Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes.As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13.In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP Nancy Université, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.

ABSTRACT
Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

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Test of the activity of Pab aPus7 and Sso aPus7 recombinant WT or variant proteins on the [α-32P]ATP-labelled P. abyssi tRNAAsp(GUC). Enzymatic tests were performed as described in the legend to Figure 5, except that we used point-mutated variants of the enzymes, Pab aPus7 K19I, R78A/H79N, and with the three R78A, H79N and K19I mutations and Sso aPus7 I127K, A90R/N91H and with the three I127K, A90R and N91H mutations. Incubations with the enzymes were performed both at 55°C (A) and at 80°C (B) and the molar amounts of Ψ residue formed per mole of tRNAAsp are given in a histogram. Error bars correspond to the standard deviations in three independent experiments.
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Figure 7: Test of the activity of Pab aPus7 and Sso aPus7 recombinant WT or variant proteins on the [α-32P]ATP-labelled P. abyssi tRNAAsp(GUC). Enzymatic tests were performed as described in the legend to Figure 5, except that we used point-mutated variants of the enzymes, Pab aPus7 K19I, R78A/H79N, and with the three R78A, H79N and K19I mutations and Sso aPus7 I127K, A90R/N91H and with the three I127K, A90R and N91H mutations. Incubations with the enzymes were performed both at 55°C (A) and at 80°C (B) and the molar amounts of Ψ residue formed per mole of tRNAAsp are given in a histogram. Error bars correspond to the standard deviations in three independent experiments.

Mentions: We tested which of the observed amino acid substitutions in motifs II and IIIa of Sso aPus7 might explain its lower activity compared to Pab aPus7. In order to dissociate the effect of amino acid substitutions in motif IIIa from amino acid substitutions in motif II, the K27I substitution found in motif IIIa of the Sso aPus7 was transferred into the Pab aPus7 enzyme by site-directed mutagenesis (variant Pab K19I). We also transferred the R90A/H91N sequence found in motif II of Sso aPus7 in Pab aPus7 (variant R78A/H79N). Finally, we produced a Pab aPus7 variant mutated in both motifs (K19I + R78A/H79N). Conversely, we tested whether we could restore some activity in Sso aPus7 by individual I27K substitution in motif IIIa, double A90R and N91H substitutions in motif II or by combination of these three amino acid substitutions. Variant Pab aPus7 and Sso aPus7 were produced as recombinant proteins in E. coli and purified using their His6-tag sequence. As WT Pab aPus7 modifies only position 13 in Pab tRNAAsp, this substrate was used to test the activity of the variant proteins. As above, each test of activity was first done at 55°C in order to preserve the RNA structure. The mean values obtained in three independent experiments are shown in Figure 7. Both the K19I and R78A/H79N substitutions in Pab aPus7 decreased the activity at position 13 in the Pab tRNAAsp at 55°C (Figure 7A), and combination of the three amino acid substitutions completely abolished this activity. Restoration of an RH pair in motif II of Sso aPus7 had almost no effect on its activity at 55°C, whereas restoration of the conserved basic residue in motif IIIa (I27K variant) allowed a significant increase of U to Ψ conversion (Figure 7A). However, the combination of all three substitutions had no positive effect. Interestingly, at 80°C (Figure 7B), the individual K19I or R78A/H79N substitutions in the Pab aPus7 had almost no negative effect on its activity towards the Pab tRNATyr(GUA). However, their combination nearly abolished this activity. As described above (Figure 5C), the WT Sso aPus7 showed some activity on the Pab tRNAAsp(GUA) at 80°C. The I27K substitution in this enzyme noticeably increased this activity (0.71 mol of Ψ/mol of tRNA, against 0.45 for the WT enzyme). However, here again the A78R and N79H substitutions had no positive effect on this activity.Figure 7.


Deficiency of the tRNATyr:Psi 35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery.

Muller S, Urban A, Hecker A, Leclerc F, Branlant C, Motorin Y - Nucleic Acids Res. (2009)

Test of the activity of Pab aPus7 and Sso aPus7 recombinant WT or variant proteins on the [α-32P]ATP-labelled P. abyssi tRNAAsp(GUC). Enzymatic tests were performed as described in the legend to Figure 5, except that we used point-mutated variants of the enzymes, Pab aPus7 K19I, R78A/H79N, and with the three R78A, H79N and K19I mutations and Sso aPus7 I127K, A90R/N91H and with the three I127K, A90R and N91H mutations. Incubations with the enzymes were performed both at 55°C (A) and at 80°C (B) and the molar amounts of Ψ residue formed per mole of tRNAAsp are given in a histogram. Error bars correspond to the standard deviations in three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651775&req=5

Figure 7: Test of the activity of Pab aPus7 and Sso aPus7 recombinant WT or variant proteins on the [α-32P]ATP-labelled P. abyssi tRNAAsp(GUC). Enzymatic tests were performed as described in the legend to Figure 5, except that we used point-mutated variants of the enzymes, Pab aPus7 K19I, R78A/H79N, and with the three R78A, H79N and K19I mutations and Sso aPus7 I127K, A90R/N91H and with the three I127K, A90R and N91H mutations. Incubations with the enzymes were performed both at 55°C (A) and at 80°C (B) and the molar amounts of Ψ residue formed per mole of tRNAAsp are given in a histogram. Error bars correspond to the standard deviations in three independent experiments.
Mentions: We tested which of the observed amino acid substitutions in motifs II and IIIa of Sso aPus7 might explain its lower activity compared to Pab aPus7. In order to dissociate the effect of amino acid substitutions in motif IIIa from amino acid substitutions in motif II, the K27I substitution found in motif IIIa of the Sso aPus7 was transferred into the Pab aPus7 enzyme by site-directed mutagenesis (variant Pab K19I). We also transferred the R90A/H91N sequence found in motif II of Sso aPus7 in Pab aPus7 (variant R78A/H79N). Finally, we produced a Pab aPus7 variant mutated in both motifs (K19I + R78A/H79N). Conversely, we tested whether we could restore some activity in Sso aPus7 by individual I27K substitution in motif IIIa, double A90R and N91H substitutions in motif II or by combination of these three amino acid substitutions. Variant Pab aPus7 and Sso aPus7 were produced as recombinant proteins in E. coli and purified using their His6-tag sequence. As WT Pab aPus7 modifies only position 13 in Pab tRNAAsp, this substrate was used to test the activity of the variant proteins. As above, each test of activity was first done at 55°C in order to preserve the RNA structure. The mean values obtained in three independent experiments are shown in Figure 7. Both the K19I and R78A/H79N substitutions in Pab aPus7 decreased the activity at position 13 in the Pab tRNAAsp at 55°C (Figure 7A), and combination of the three amino acid substitutions completely abolished this activity. Restoration of an RH pair in motif II of Sso aPus7 had almost no effect on its activity at 55°C, whereas restoration of the conserved basic residue in motif IIIa (I27K variant) allowed a significant increase of U to Ψ conversion (Figure 7A). However, the combination of all three substitutions had no positive effect. Interestingly, at 80°C (Figure 7B), the individual K19I or R78A/H79N substitutions in the Pab aPus7 had almost no negative effect on its activity towards the Pab tRNATyr(GUA). However, their combination nearly abolished this activity. As described above (Figure 5C), the WT Sso aPus7 showed some activity on the Pab tRNAAsp(GUA) at 80°C. The I27K substitution in this enzyme noticeably increased this activity (0.71 mol of Ψ/mol of tRNA, against 0.45 for the WT enzyme). However, here again the A78R and N79H substitutions had no positive effect on this activity.Figure 7.

Bottom Line: Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes.As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13.In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP Nancy Université, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.

ABSTRACT
Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

Show MeSH
Related in: MedlinePlus