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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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Aminoacylation kinetics for tRNAAsp and tRNAAsn with Hp ND-AspRS: a dual response to substrates. Experiments were conducted in 15% glycerol at 4°C for tRNAAsp (black diamonds) and tRNAAsn (gray triangles). Results show that tRNAAsp and tRNAAsn are aminoacylated differently. In case of tRNAAsp, the first cycle (1 Asp-tRNA formed) has the same rate as the subsequent ones (0.04 s−1). However, in case of tRNAAsn, the first cycle (0.04 s−1), which is equivalent to that for tRNAAsp, is significantly faster than the subsequent ones (0.0033 s−1). Extrapolation of this slower phase at t0 points to the formation of 1 Asp-tRNAAsn per ND-AspRS active site. This pattern suggests a rate-limiting step which would be the release of Asp-tRNAAsn. SE was <5% on all values.
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gks167-F3: Aminoacylation kinetics for tRNAAsp and tRNAAsn with Hp ND-AspRS: a dual response to substrates. Experiments were conducted in 15% glycerol at 4°C for tRNAAsp (black diamonds) and tRNAAsn (gray triangles). Results show that tRNAAsp and tRNAAsn are aminoacylated differently. In case of tRNAAsp, the first cycle (1 Asp-tRNA formed) has the same rate as the subsequent ones (0.04 s−1). However, in case of tRNAAsn, the first cycle (0.04 s−1), which is equivalent to that for tRNAAsp, is significantly faster than the subsequent ones (0.0033 s−1). Extrapolation of this slower phase at t0 points to the formation of 1 Asp-tRNAAsn per ND-AspRS active site. This pattern suggests a rate-limiting step which would be the release of Asp-tRNAAsn. SE was <5% on all values.

Mentions: Table 1 summarizes the steady-state kinetic parameters of Hp ND-AspRS for aminoacylation of tRNAAsp and tRNAAsn. The enzyme aspartylates tRNAAsp 2.5-fold faster than tRNAAsn (0.33 and 0.14 s−1), as reported previously (32). However, pre-steady-state kinetics conducted under conditions that allowed an examination of the first catalytic cycles of the enzyme (4°C and in the presence of 10 or 15% of glycerol) revealed intriguing differences in aminoacylation of both tRNAs since in contrast to tRNAAsp the charging kinetics of tRNAAsn were biphasic. Aminoacylation of tRNAAsp remains linear after completion of the first catalytic cycle. Thus, this first cycle occurs with the same rate as subsequent ones (0.041 s−1) (Figure 3). Since ATP-PPi exchange rate is significantly faster than tRNA charging (15.9 and 0.33 s−1 at 37°C), the steady-state rate of tRNAAsp aminoacylation is dictated by the rate of transfer of activated Asp onto tRNAAsp. Surprisingly, when tRNAAsn is used as a substrate, biphasic kinetics arise, which exhibit a burst of Asp-tRNAAsn formation (∼0.04 s−1) followed by a significantly slower linear phase (0.0033 s−1) (Figure 3). Extrapolation of the linear phase at t0 points to formation of one Asp-tRNAAsn per ND-AspRS active site during the fast phase (Figure 3) and suggests that the first tRNAAsn is aspartylated significantly faster than those following. Interestingly, the first tRNAAsn is aminoacylated with a rate equivalent to that observed for tRNAAsp (Figure 3). Burst and steady-state rate values are both dependent on enzyme concentration (Supplementary Figure S1A). In the slow phase, the steady-state rate of tRNAAsn charging increases with the pH but not significantly with the ionic strength (Supplementary Figure S1B). This kinetic behavior is consistent with the release of Asp-tRNAAsn being the rate-limiting step at the steady-state of the reaction (40). The slow dissociation of the Asp-tRNAAsn product agrees with the absence of detectable hydrolysis of its ester bond in the presence of ND-AspRS (28).Figure 3.


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)

Aminoacylation kinetics for tRNAAsp and tRNAAsn with Hp ND-AspRS: a dual response to substrates. Experiments were conducted in 15% glycerol at 4°C for tRNAAsp (black diamonds) and tRNAAsn (gray triangles). Results show that tRNAAsp and tRNAAsn are aminoacylated differently. In case of tRNAAsp, the first cycle (1 Asp-tRNA formed) has the same rate as the subsequent ones (0.04 s−1). However, in case of tRNAAsn, the first cycle (0.04 s−1), which is equivalent to that for tRNAAsp, is significantly faster than the subsequent ones (0.0033 s−1). Extrapolation of this slower phase at t0 points to the formation of 1 Asp-tRNAAsn per ND-AspRS active site. This pattern suggests a rate-limiting step which would be the release of Asp-tRNAAsn. SE was <5% on all values.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks167-F3: Aminoacylation kinetics for tRNAAsp and tRNAAsn with Hp ND-AspRS: a dual response to substrates. Experiments were conducted in 15% glycerol at 4°C for tRNAAsp (black diamonds) and tRNAAsn (gray triangles). Results show that tRNAAsp and tRNAAsn are aminoacylated differently. In case of tRNAAsp, the first cycle (1 Asp-tRNA formed) has the same rate as the subsequent ones (0.04 s−1). However, in case of tRNAAsn, the first cycle (0.04 s−1), which is equivalent to that for tRNAAsp, is significantly faster than the subsequent ones (0.0033 s−1). Extrapolation of this slower phase at t0 points to the formation of 1 Asp-tRNAAsn per ND-AspRS active site. This pattern suggests a rate-limiting step which would be the release of Asp-tRNAAsn. SE was <5% on all values.
Mentions: Table 1 summarizes the steady-state kinetic parameters of Hp ND-AspRS for aminoacylation of tRNAAsp and tRNAAsn. The enzyme aspartylates tRNAAsp 2.5-fold faster than tRNAAsn (0.33 and 0.14 s−1), as reported previously (32). However, pre-steady-state kinetics conducted under conditions that allowed an examination of the first catalytic cycles of the enzyme (4°C and in the presence of 10 or 15% of glycerol) revealed intriguing differences in aminoacylation of both tRNAs since in contrast to tRNAAsp the charging kinetics of tRNAAsn were biphasic. Aminoacylation of tRNAAsp remains linear after completion of the first catalytic cycle. Thus, this first cycle occurs with the same rate as subsequent ones (0.041 s−1) (Figure 3). Since ATP-PPi exchange rate is significantly faster than tRNA charging (15.9 and 0.33 s−1 at 37°C), the steady-state rate of tRNAAsp aminoacylation is dictated by the rate of transfer of activated Asp onto tRNAAsp. Surprisingly, when tRNAAsn is used as a substrate, biphasic kinetics arise, which exhibit a burst of Asp-tRNAAsn formation (∼0.04 s−1) followed by a significantly slower linear phase (0.0033 s−1) (Figure 3). Extrapolation of the linear phase at t0 points to formation of one Asp-tRNAAsn per ND-AspRS active site during the fast phase (Figure 3) and suggests that the first tRNAAsn is aspartylated significantly faster than those following. Interestingly, the first tRNAAsn is aminoacylated with a rate equivalent to that observed for tRNAAsp (Figure 3). Burst and steady-state rate values are both dependent on enzyme concentration (Supplementary Figure S1A). In the slow phase, the steady-state rate of tRNAAsn charging increases with the pH but not significantly with the ionic strength (Supplementary Figure S1B). This kinetic behavior is consistent with the release of Asp-tRNAAsn being the rate-limiting step at the steady-state of the reaction (40). The slow dissociation of the Asp-tRNAAsn product agrees with the absence of detectable hydrolysis of its ester bond in the presence of ND-AspRS (28).Figure 3.

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