Published online 22 February 2012 Nucleic Acids Research, 2012, Vol. 40, No. 11 4965–4976 doi:10.1093/nar/gks167 The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code Fre´ de´ ric Fischer1, Jonathan L. Huot2, Bernard Lorber1, Guillaume Diss1, Tamara L. Hendrickson 3 , Hubert D. Becker1, Jacques Lapointe2,* and Daniel Kern1,* 1Institut de Biologie Mole´ culaire et Cellulaire, UPR 9002 du CNRS, Architecture et Re´ activite´ de l’ARN, Universite´ de Strasbourg, 15 rue Rene´ Descartes, 67084 Strasbourg Cedex, France, 2De´ partement de Biochimie, de Microbiologie et de Bio-informatique, PROTEO, Universite´ Laval, 1045 av. de la Me´ decine, Que´ bec (Que´ bec), Canada G1V 0A6 and 3Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA Received November 1, 2011; Revised January 31, 2012; Accepted February 1, 2012 ABSTRACT Helicobacter pylori catalyzes Asn-tRNAAsn forma- tion by use of the indirect pathway that involves charging of Asp onto tRNAAsn by a non-discri- minating 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 differ- ent 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) tRNAAsn binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNAAsn is bound by ND-AspRS which releases the Asp-tRNAAsn product much slower than the cognate Asp-tRNAAsp; this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNAAsn 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. INTRODUCTION Accurate expression of genetic information is essential to all living organisms. Integrity of the message encoded within a gene is safeguarded by mechanisms that cells have evolved at each step of gene expression. In protein synthesis, tRNA aminoacylation is a crucial step where each tRNA species is bound to its cognate amino acid in reactions catalyzed by the aminoacyl-tRNA synthetases (aaRSs) (1,2). The amino acids are ﬁrst activated as aminoacyl AMPs and then transferred onto their cognate tRNAs to synthesize the ﬁnal aminoacyl-tRNA products. The latter are then carried to the ribosome by an elongation factor where they are used to decode the codons on the mRNA. Fidelity of translation depends on accurate tRNA aminoacylation, i.e. correct matching of an amino acid to its set of isoacceptor tRNAs. This coupling is determined by the existence of a particular aaRS for each amino acid/tRNA pair. The capacity to select homologous substrates is conferred by active site geometry and by correction processes that are used by aaRSs when the wrong aminoacyl-tRNAs are formed *To whom correspondence should be addressed. Tel: +33 3 88 41 70 92; Fax: +33 3 88 60 22 18; Email: d.kern@ibmc-cnrs.unistra.fr Correspondence may also be addressed to Jacques Lapointe. Tel: +1 418 656 2131 (Extn 3411); Fax: +1 418 656 3664; Email: jacques.lapointe@bcm.ulaval.ca The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.