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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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Related in: MedlinePlus

Formation of Asn-tRNAAsn in H. pylori. (A) The major pathway used in H. pylori (the main ‘Asn-transamidosome pathway’) starts with (1) the formation of the tRNPC, linking GatCAB and tRNAAsn, onto which (2) ND-AspRS can then dock to form the Asn-transamidosome. In this ribonucleoprotein, tRNAAsn gets aspartylated more efficiently (3), and the aspartylated CCA-end of tRNAAsn shifts from the ND-AspRS active site to the GatCAB phosphorylation/amidation active site. Asn-tRNAAsn biosynthesis proceeds thanks to amide donor hydrolysis (here free Gln) to provide the ammonia moiety necessary for transamidation (4) (28). When Asn-tRNAAsn is formed, it is released together with ND-AspRS, and used in protein synthesis (5). (B) When GatCAB is not in sufficient amounts to produce enough tRNPC, free tRNAAsn binds to ND-AspRS (6) and gets aminoacylated less efficiently (7). The dissociation of Asp-tRNAAsn is rate-limiting, but if Asp-tRNAAsn is released (8), GatCAB can still recognize it (9) and transamidate it (10) (28) into Asn-tRNAAsn, which can be released to fuel translation (11). (C) Since the release of Asp-tRNAAsn from ND-AspRS is rate-limiting, Asp-tRNAAsn remains within the ND-AspRS active site for longer times, enabling free GatCAB to bind to the ND-AspRS/Asp-tRNAAsn complex (12) to form an Asn-transamidosome, in which Asn-tRNAAsn can be synthesized through transamidation (4) rejoining the main Asn-transamidosome cycle. This latter mechanism can be qualified as a ‘rescue mechanism’. The table summarizes the different pathway characteristics. The aspartylation and transamidation steps are highlighted to stress the fact that the Asn-transamidosome pathway couples both reactions, while they are sequential when GatCAB is absent or when the tRNPC is not involved.
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gks167-SCH1: Formation of Asn-tRNAAsn in H. pylori. (A) The major pathway used in H. pylori (the main ‘Asn-transamidosome pathway’) starts with (1) the formation of the tRNPC, linking GatCAB and tRNAAsn, onto which (2) ND-AspRS can then dock to form the Asn-transamidosome. In this ribonucleoprotein, tRNAAsn gets aspartylated more efficiently (3), and the aspartylated CCA-end of tRNAAsn shifts from the ND-AspRS active site to the GatCAB phosphorylation/amidation active site. Asn-tRNAAsn biosynthesis proceeds thanks to amide donor hydrolysis (here free Gln) to provide the ammonia moiety necessary for transamidation (4) (28). When Asn-tRNAAsn is formed, it is released together with ND-AspRS, and used in protein synthesis (5). (B) When GatCAB is not in sufficient amounts to produce enough tRNPC, free tRNAAsn binds to ND-AspRS (6) and gets aminoacylated less efficiently (7). The dissociation of Asp-tRNAAsn is rate-limiting, but if Asp-tRNAAsn is released (8), GatCAB can still recognize it (9) and transamidate it (10) (28) into Asn-tRNAAsn, which can be released to fuel translation (11). (C) Since the release of Asp-tRNAAsn from ND-AspRS is rate-limiting, Asp-tRNAAsn remains within the ND-AspRS active site for longer times, enabling free GatCAB to bind to the ND-AspRS/Asp-tRNAAsn complex (12) to form an Asn-transamidosome, in which Asn-tRNAAsn can be synthesized through transamidation (4) rejoining the main Asn-transamidosome cycle. This latter mechanism can be qualified as a ‘rescue mechanism’. The table summarizes the different pathway characteristics. The aspartylation and transamidation steps are highlighted to stress the fact that the Asn-transamidosome pathway couples both reactions, while they are sequential when GatCAB is absent or when the tRNPC is not involved.


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)

Formation of Asn-tRNAAsn in H. pylori. (A) The major pathway used in H. pylori (the main ‘Asn-transamidosome pathway’) starts with (1) the formation of the tRNPC, linking GatCAB and tRNAAsn, onto which (2) ND-AspRS can then dock to form the Asn-transamidosome. In this ribonucleoprotein, tRNAAsn gets aspartylated more efficiently (3), and the aspartylated CCA-end of tRNAAsn shifts from the ND-AspRS active site to the GatCAB phosphorylation/amidation active site. Asn-tRNAAsn biosynthesis proceeds thanks to amide donor hydrolysis (here free Gln) to provide the ammonia moiety necessary for transamidation (4) (28). When Asn-tRNAAsn is formed, it is released together with ND-AspRS, and used in protein synthesis (5). (B) When GatCAB is not in sufficient amounts to produce enough tRNPC, free tRNAAsn binds to ND-AspRS (6) and gets aminoacylated less efficiently (7). The dissociation of Asp-tRNAAsn is rate-limiting, but if Asp-tRNAAsn is released (8), GatCAB can still recognize it (9) and transamidate it (10) (28) into Asn-tRNAAsn, which can be released to fuel translation (11). (C) Since the release of Asp-tRNAAsn from ND-AspRS is rate-limiting, Asp-tRNAAsn remains within the ND-AspRS active site for longer times, enabling free GatCAB to bind to the ND-AspRS/Asp-tRNAAsn complex (12) to form an Asn-transamidosome, in which Asn-tRNAAsn can be synthesized through transamidation (4) rejoining the main Asn-transamidosome cycle. This latter mechanism can be qualified as a ‘rescue mechanism’. The table summarizes the different pathway characteristics. The aspartylation and transamidation steps are highlighted to stress the fact that the Asn-transamidosome pathway couples both reactions, while they are sequential when GatCAB is absent or when the tRNPC is not involved.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks167-SCH1: Formation of Asn-tRNAAsn in H. pylori. (A) The major pathway used in H. pylori (the main ‘Asn-transamidosome pathway’) starts with (1) the formation of the tRNPC, linking GatCAB and tRNAAsn, onto which (2) ND-AspRS can then dock to form the Asn-transamidosome. In this ribonucleoprotein, tRNAAsn gets aspartylated more efficiently (3), and the aspartylated CCA-end of tRNAAsn shifts from the ND-AspRS active site to the GatCAB phosphorylation/amidation active site. Asn-tRNAAsn biosynthesis proceeds thanks to amide donor hydrolysis (here free Gln) to provide the ammonia moiety necessary for transamidation (4) (28). When Asn-tRNAAsn is formed, it is released together with ND-AspRS, and used in protein synthesis (5). (B) When GatCAB is not in sufficient amounts to produce enough tRNPC, free tRNAAsn binds to ND-AspRS (6) and gets aminoacylated less efficiently (7). The dissociation of Asp-tRNAAsn is rate-limiting, but if Asp-tRNAAsn is released (8), GatCAB can still recognize it (9) and transamidate it (10) (28) into Asn-tRNAAsn, which can be released to fuel translation (11). (C) Since the release of Asp-tRNAAsn from ND-AspRS is rate-limiting, Asp-tRNAAsn remains within the ND-AspRS active site for longer times, enabling free GatCAB to bind to the ND-AspRS/Asp-tRNAAsn complex (12) to form an Asn-transamidosome, in which Asn-tRNAAsn can be synthesized through transamidation (4) rejoining the main Asn-transamidosome cycle. This latter mechanism can be qualified as a ‘rescue mechanism’. The table summarizes the different pathway characteristics. The aspartylation and transamidation steps are highlighted to stress the fact that the Asn-transamidosome pathway couples both reactions, while they are sequential when GatCAB is absent or when the tRNPC is not involved.
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