Limits...
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.

Show MeSH
Size-exclusion chromatography shows formation of a transamidosome complex by ND-AspRS, GatCAB and tRNAAsn. (A) Comparison of isolated ND-AspRS and ND-AspRS/tRNAAsp and ND-AspRS/tRNAAsn complexes. The observed association enabled an estimation of the KD value for binding of each tRNA to ND-AspRS (see ‘Materials and Methods’ section). KD values for the binding to ND-AspRS are 7.9 and 21.4 µM for tRNAAsp and tRNAAsn, respectively. (B) Comparison between free GatCAB and GatCAB/tRNAAsn complexes. The KD value for this association is 2.1 µM. (C) Elution profile of a mixture containing GatCAB, ND-AspRS and tRNAAsn. (D) SDS–PAGE profile of a SEC fraction (lane 3) compared to GatCAB alone (lane 1) and ND-AspRS alone (lane 2). (E) TLC plate demonstrating that the complex eluted in the first peak (termed ‘isolated’) is able to produce Asn-tRNAAsn alone, or when supplemented with excess tRNAAsn (termed ‘+2 µM tRNA’), compared to a negative control where GatCAB was omitted. All partners were mixed to a final concentration of 20 µM each. All KD values determined varied within 10%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3367201&req=5

gks167-F1: Size-exclusion chromatography shows formation of a transamidosome complex by ND-AspRS, GatCAB and tRNAAsn. (A) Comparison of isolated ND-AspRS and ND-AspRS/tRNAAsp and ND-AspRS/tRNAAsn complexes. The observed association enabled an estimation of the KD value for binding of each tRNA to ND-AspRS (see ‘Materials and Methods’ section). KD values for the binding to ND-AspRS are 7.9 and 21.4 µM for tRNAAsp and tRNAAsn, respectively. (B) Comparison between free GatCAB and GatCAB/tRNAAsn complexes. The KD value for this association is 2.1 µM. (C) Elution profile of a mixture containing GatCAB, ND-AspRS and tRNAAsn. (D) SDS–PAGE profile of a SEC fraction (lane 3) compared to GatCAB alone (lane 1) and ND-AspRS alone (lane 2). (E) TLC plate demonstrating that the complex eluted in the first peak (termed ‘isolated’) is able to produce Asn-tRNAAsn alone, or when supplemented with excess tRNAAsn (termed ‘+2 µM tRNA’), compared to a negative control where GatCAB was omitted. All partners were mixed to a final concentration of 20 µM each. All KD values determined varied within 10%.

Mentions: Association of ND-AspRS, tRNAAsn and GatCAB from T. thermophilus leads to a stable ternary complex named Asn-transamidosome (21). Gel-filtration and DLS experiments conducted with the H. pylori partners show that the ND-AspRS can bind both tRNAAsp and tRNAAsn but with different affinities (KD values of 7.9 and 21.4 µM respectively, Figure 1A) and that GatCAB can form a much more stable binary complex with tRNAAsn than ND-AspRS (KD value of 2.1 µM, Figure 1B). These divergent KD values differ from those obtained using the T. thermophilus partners, where tRNAAsn bound to ND-AspRS with higher affinity than to GatCAB (21). The asymmetric KD values determined with the H. pylori system were confirmed by DLS (Figure 2). Indeed, a higher-sized particle was clearly seen in the presence of ND-AspRS and tRNAAsp (12.6 nm), compared to free ND-AspRS and tRNA (10.9 and 4.9 nm, respectively) but not when ND-AspRS and tRNAAsn were mixed (Figure 2, lanes 1–4) (10.9 nm). Similarly, we detected an association between GatCAB and tRNAAsn (10.9 nm) compared to their isolated counterparts (9.4 and 4.9 nm, respectively) (Figure 2, lanes 5 and 6). No association of the protein partners in the absence of tRNA was detected by either technique (data not shown). Finally, when the three partners were mixed, a new ribonucleoprotein (RNP) of significantly higher size (13.5 nm) appeared (Figure 2, lane 7). Isolation of this complex by gel-filtration (Figure 1C) and analysis of its components by SDS–PAGE (Figure 1D) revealed the presence of ND-AspRS, the three GatCAB subunits and tRNAAsn. Functional analysis confirmed that it was fully able to synthesize Asn-tRNAAsn in the presence of free Asp, ATP and Gln (Figure 1E). Because the association of the protein partners is tRNAAsn-dependent, this complex constitutes a bona fide Asn-transamidosome according to previous arguments (21).Figure 1.


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)

Size-exclusion chromatography shows formation of a transamidosome complex by ND-AspRS, GatCAB and tRNAAsn. (A) Comparison of isolated ND-AspRS and ND-AspRS/tRNAAsp and ND-AspRS/tRNAAsn complexes. The observed association enabled an estimation of the KD value for binding of each tRNA to ND-AspRS (see ‘Materials and Methods’ section). KD values for the binding to ND-AspRS are 7.9 and 21.4 µM for tRNAAsp and tRNAAsn, respectively. (B) Comparison between free GatCAB and GatCAB/tRNAAsn complexes. The KD value for this association is 2.1 µM. (C) Elution profile of a mixture containing GatCAB, ND-AspRS and tRNAAsn. (D) SDS–PAGE profile of a SEC fraction (lane 3) compared to GatCAB alone (lane 1) and ND-AspRS alone (lane 2). (E) TLC plate demonstrating that the complex eluted in the first peak (termed ‘isolated’) is able to produce Asn-tRNAAsn alone, or when supplemented with excess tRNAAsn (termed ‘+2 µM tRNA’), compared to a negative control where GatCAB was omitted. All partners were mixed to a final concentration of 20 µM each. All KD values determined varied within 10%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks167-F1: Size-exclusion chromatography shows formation of a transamidosome complex by ND-AspRS, GatCAB and tRNAAsn. (A) Comparison of isolated ND-AspRS and ND-AspRS/tRNAAsp and ND-AspRS/tRNAAsn complexes. The observed association enabled an estimation of the KD value for binding of each tRNA to ND-AspRS (see ‘Materials and Methods’ section). KD values for the binding to ND-AspRS are 7.9 and 21.4 µM for tRNAAsp and tRNAAsn, respectively. (B) Comparison between free GatCAB and GatCAB/tRNAAsn complexes. The KD value for this association is 2.1 µM. (C) Elution profile of a mixture containing GatCAB, ND-AspRS and tRNAAsn. (D) SDS–PAGE profile of a SEC fraction (lane 3) compared to GatCAB alone (lane 1) and ND-AspRS alone (lane 2). (E) TLC plate demonstrating that the complex eluted in the first peak (termed ‘isolated’) is able to produce Asn-tRNAAsn alone, or when supplemented with excess tRNAAsn (termed ‘+2 µM tRNA’), compared to a negative control where GatCAB was omitted. All partners were mixed to a final concentration of 20 µM each. All KD values determined varied within 10%.
Mentions: Association of ND-AspRS, tRNAAsn and GatCAB from T. thermophilus leads to a stable ternary complex named Asn-transamidosome (21). Gel-filtration and DLS experiments conducted with the H. pylori partners show that the ND-AspRS can bind both tRNAAsp and tRNAAsn but with different affinities (KD values of 7.9 and 21.4 µM respectively, Figure 1A) and that GatCAB can form a much more stable binary complex with tRNAAsn than ND-AspRS (KD value of 2.1 µM, Figure 1B). These divergent KD values differ from those obtained using the T. thermophilus partners, where tRNAAsn bound to ND-AspRS with higher affinity than to GatCAB (21). The asymmetric KD values determined with the H. pylori system were confirmed by DLS (Figure 2). Indeed, a higher-sized particle was clearly seen in the presence of ND-AspRS and tRNAAsp (12.6 nm), compared to free ND-AspRS and tRNA (10.9 and 4.9 nm, respectively) but not when ND-AspRS and tRNAAsn were mixed (Figure 2, lanes 1–4) (10.9 nm). Similarly, we detected an association between GatCAB and tRNAAsn (10.9 nm) compared to their isolated counterparts (9.4 and 4.9 nm, respectively) (Figure 2, lanes 5 and 6). No association of the protein partners in the absence of tRNA was detected by either technique (data not shown). Finally, when the three partners were mixed, a new ribonucleoprotein (RNP) of significantly higher size (13.5 nm) appeared (Figure 2, lane 7). Isolation of this complex by gel-filtration (Figure 1C) and analysis of its components by SDS–PAGE (Figure 1D) revealed the presence of ND-AspRS, the three GatCAB subunits and tRNAAsn. Functional analysis confirmed that it was fully able to synthesize Asn-tRNAAsn in the presence of free Asp, ATP and Gln (Figure 1E). Because the association of the protein partners is tRNAAsn-dependent, this complex constitutes a bona fide Asn-transamidosome according to previous arguments (21).Figure 1.

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