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The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code.

Fischer F, Huot JL, Lorber B, Diss G, Hendrickson TL, Becker HD, Lapointe J, Kern D - Nucleic Acids Res. (2012)

Bottom Line: We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate.Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS.These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire et Cellulaire, UPR 9002 du CNRS, Architecture et Réactivité de l'ARN, Université de Strasbourg, 15 rue René Descartes, 67084 Strasbourg Cedex, France.

ABSTRACT
Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

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Effects of GatCAB on aminoacylation of tRNAAsn using Hp ND-AspRS versus Ec D-AspRS. The graph shows the effect of increasing GatCAB concentrations on initial rates of both AspRSs in the presence of saturating tRNAAsn concentration. Results (v) were normalized using the steady-state rate value of the corresponding AspRS in absence of GatCAB (v0). Concentration of the GatCAB/tRNAAsn complex increases together with GatCAB concentration. (A) GatCAB increases the steady-state rate of Hp ND-AspRS when tRNAAsn is present (gray triangles), but not when tRNAAsp is used (inset). Error bars represent the SD from three independent experiments. (B) GatCAB decreases the steady-state rate of tRNAAsn misacylation with Ec D-AspRS.
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gks167-F4: Effects of GatCAB on aminoacylation of tRNAAsn using Hp ND-AspRS versus Ec D-AspRS. The graph shows the effect of increasing GatCAB concentrations on initial rates of both AspRSs in the presence of saturating tRNAAsn concentration. Results (v) were normalized using the steady-state rate value of the corresponding AspRS in absence of GatCAB (v0). Concentration of the GatCAB/tRNAAsn complex increases together with GatCAB concentration. (A) GatCAB increases the steady-state rate of Hp ND-AspRS when tRNAAsn is present (gray triangles), but not when tRNAAsp is used (inset). Error bars represent the SD from three independent experiments. (B) GatCAB decreases the steady-state rate of tRNAAsn misacylation with Ec D-AspRS.

Mentions: Table 1 shows the effect of GatCAB on kinetic constants of Hp ND-AspRS for tRNAAsn and tRNAAsp aspartylation. The addition of GatCAB increases the steady-state rate of Asp-tRNAAsn formation according to a hyperbolic curve (Figure 4A), but has no effect on the steady-state rate of tRNAAsp charging (Figure 4A, inset and Table 1), indicating that this phenomenon is specific to tRNAAsn. When saturating concentrations of GatCAB are reached, the rate of tRNAAsn aspartylation increases 1.8-fold (0.14–0.25 s−1) and nearly fits that of tRNAAsp (0.33 s−1, Table 1), while the KM value of ND-AspRS for tRNAAsn increases 3.8-fold, leading to an increase in the overall charging efficiency (kcat/KM) of 1.8-fold (Table 1). Thus, in the presence of GatCAB, aminoacylation of tRNAAsn occurs with a similar efficiency as that of tRNAAsp (kcat/KM, respectively of 0.27 and 0.30 µM−1 s−1). Pre-steady-state kinetics reveal that this GatCAB-mediated effect originates in an increase in the rate of aminoacylation during the slow phase (0.0033–0.012 s−1, Figure 5A and B). The rate of the fast phase does not seem to be affected (Figure 5B). Considering that tRNAAsn binds GatCAB and ND-AspRS with KD values of 2.1 and 21.4 µM, respectively (Figure 1), and that aspartylation of tRNAAsn is more efficient when it is ‘labeled’ with or ‘presented’ by GatCAB (Table 1), the GatCAB/tRNAAsn complex may constitute a better substrate for misacylation by the ND-AspRS. We named this complex the tRNAAsn-presentation complex (tRNPC). Since this complex is non-productive in absence of ND-AspRS and can also be a substrate, tRNPC can be considered as a bona fide transfer ribonucleoprotein (tRNP).Figure 4.


The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code.

Fischer F, Huot JL, Lorber B, Diss G, Hendrickson TL, Becker HD, Lapointe J, Kern D - Nucleic Acids Res. (2012)

Effects of GatCAB on aminoacylation of tRNAAsn using Hp ND-AspRS versus Ec D-AspRS. The graph shows the effect of increasing GatCAB concentrations on initial rates of both AspRSs in the presence of saturating tRNAAsn concentration. Results (v) were normalized using the steady-state rate value of the corresponding AspRS in absence of GatCAB (v0). Concentration of the GatCAB/tRNAAsn complex increases together with GatCAB concentration. (A) GatCAB increases the steady-state rate of Hp ND-AspRS when tRNAAsn is present (gray triangles), but not when tRNAAsp is used (inset). Error bars represent the SD from three independent experiments. (B) GatCAB decreases the steady-state rate of tRNAAsn misacylation with Ec D-AspRS.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367201&req=5

gks167-F4: Effects of GatCAB on aminoacylation of tRNAAsn using Hp ND-AspRS versus Ec D-AspRS. The graph shows the effect of increasing GatCAB concentrations on initial rates of both AspRSs in the presence of saturating tRNAAsn concentration. Results (v) were normalized using the steady-state rate value of the corresponding AspRS in absence of GatCAB (v0). Concentration of the GatCAB/tRNAAsn complex increases together with GatCAB concentration. (A) GatCAB increases the steady-state rate of Hp ND-AspRS when tRNAAsn is present (gray triangles), but not when tRNAAsp is used (inset). Error bars represent the SD from three independent experiments. (B) GatCAB decreases the steady-state rate of tRNAAsn misacylation with Ec D-AspRS.
Mentions: Table 1 shows the effect of GatCAB on kinetic constants of Hp ND-AspRS for tRNAAsn and tRNAAsp aspartylation. The addition of GatCAB increases the steady-state rate of Asp-tRNAAsn formation according to a hyperbolic curve (Figure 4A), but has no effect on the steady-state rate of tRNAAsp charging (Figure 4A, inset and Table 1), indicating that this phenomenon is specific to tRNAAsn. When saturating concentrations of GatCAB are reached, the rate of tRNAAsn aspartylation increases 1.8-fold (0.14–0.25 s−1) and nearly fits that of tRNAAsp (0.33 s−1, Table 1), while the KM value of ND-AspRS for tRNAAsn increases 3.8-fold, leading to an increase in the overall charging efficiency (kcat/KM) of 1.8-fold (Table 1). Thus, in the presence of GatCAB, aminoacylation of tRNAAsn occurs with a similar efficiency as that of tRNAAsp (kcat/KM, respectively of 0.27 and 0.30 µM−1 s−1). Pre-steady-state kinetics reveal that this GatCAB-mediated effect originates in an increase in the rate of aminoacylation during the slow phase (0.0033–0.012 s−1, Figure 5A and B). The rate of the fast phase does not seem to be affected (Figure 5B). Considering that tRNAAsn binds GatCAB and ND-AspRS with KD values of 2.1 and 21.4 µM, respectively (Figure 1), and that aspartylation of tRNAAsn is more efficient when it is ‘labeled’ with or ‘presented’ by GatCAB (Table 1), the GatCAB/tRNAAsn complex may constitute a better substrate for misacylation by the ND-AspRS. We named this complex the tRNAAsn-presentation complex (tRNPC). Since this complex is non-productive in absence of ND-AspRS and can also be a substrate, tRNPC can be considered as a bona fide transfer ribonucleoprotein (tRNP).Figure 4.

Bottom Line: We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate.Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS.These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire et Cellulaire, UPR 9002 du CNRS, Architecture et Réactivité de l'ARN, Université de Strasbourg, 15 rue René Descartes, 67084 Strasbourg Cedex, France.

ABSTRACT
Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

Show MeSH
Related in: MedlinePlus