4966 Nucleic Acids Research, 2012, Vol. 40, No. 11 (3,4). Moreover, aaRSs are split into two classes, accord- ing to sequence/structure (5) and kinetic (6–8) properties. Members of class I bind tRNA on the minor grove side and acylate the 20-OH of the terminal adenosine whereas the members of class II approach tRNA from the major grove side and transfer the amino acid moiety on the 30-OH of the terminal adenosine. Further, it seems that most class I aaRSs display a rate-limiting step for the release of aminoacyl-tRNA, whereas the transfer of the activated amino acid onto tRNA is rate-limiting for class II aaRSs (8). Global genome analyses and biochemical data indicate that about half of prokaryotes are deprived of asparaginyl-tRNA synthetase (AsnRS), and that the vast majority lack a glutaminyl-tRNA synthetase (GlnRS). This pattern negates the old ‘dogma’ that postulated the presence of a speciﬁc aaRS for each amino acid and tRNA pair (9,10). These organisms, including mitochondria (11), all synthesize amidated aminoacyl-tRNAs thanks to an enzymatic system that includes a non-discriminating (ND) aaRS, ND-aspartyl-tRNA synthetase (ND-AspRS) and/or ND-glutamyl-tRNA synthetase (ND-GluRS), providing a misacylated aminoacyl-tRNA species (Asp-tRNAAsn or Glu-tRNAGln); these aminoacyl- tRNAs are then modiﬁed into Asn-tRNAAsn or Gln-tRNAGln through a phosphorylation/transamidation mechanism catalyzed by a tRNA-dependent amidotra- nsferase called GatCAB in bacteria (12–20). The synthesis of Asn-tRNAAsn and Gln-tRNAGln is ensured by two separate bi-enzymatic systems, consisting in functionally related enzymes brought together by a scaffold/substrate tRNA. The resulting aaRS/tRNA/GatCAB complexes are named transamidosomes. These particles vary in cohesive strength, with the Asn-transamidosome of Thermus thermophilus (21) showing the highest stability while the Gln-transamidosomes of Thermotoga maritima and Helicobacter pylori are more dynamic (22–24). Within all of these complexes, the tRNA moiety can be used either as a scaffold for the nucleoprotein to form Asn or Gln or as a substrate for their synthesis. This makes the misacylation/ transamidation system a matrix-assisted amino acid biosynthesis pathway of particular interest in H. pylori, a well-known and widespread human pathogen, which uses these indirect pathways to form Asn, Gln-tRNAGln and Asn-tRNAAsn (24–28). Although it was shown that mischarged Asp-tRNAAsn and Glu-tRNAGln species are deprived of signiﬁcant afﬁnity for the elongation factor EF-Tu (29,30), several investigations have shown that they can lead in vivo to incorporation of Asp (31,32) or Glu (33–35) in place of Asn and Gln into proteins. Overexpression of either misacylating aaRS is toxic in Escherichia coli (31–35) while in yeast a disabled amidotransferase resulted in sig- niﬁcant incorporation of Glu at Gln codons in mitochon- drial proteins (36). Asn- and Gln-transamidosomes, which control the release of misacylated intermediates, may therefore be another layer of aminoacyl-tRNA quality control. Here, we report how this quality control is achieved for Asn-tRNAAsn synthesis in H. pylori through the forma- tion of an Asn-transamidosome. Moreover, we show that ND-AspRS exhibits surprisingly sensitive kinetic properties for Asp-tRNAAsp and Asp-tRNAAsn forma- tion: fast release of Asp-tRNAAsp for translation and slow release of Asp-tRNAAsn to allow transamidosome formation by GatCAB binding. This safeguard mechan- ism resembles an editing process and may shed light on evolutionary processes that led to Asn inﬁltration into the present-day genetic code. Our results suggest that, in H. pylori, ND-AspRS and GatCAB co-evolved to generate an appropriate kinetic system able to reduce translational error level and to ensure a faithful codon reassignment for Asn. MATERIALS AND METHODS Overproduction and puriﬁcation of enzymes and tRNAs The H. pylori (Hp) ND-AspRS and GatCAB were overproduced from the cloned aspS, gatC, gatA and gatB genes as described (28,32). For protein puriﬁcation, the cells from an overnight culture were harvested by cen- trifugation at 4000g, washed in cold Tris–HCl buffer 100 mM, pH 8.0, sedimented and suspended in the cell disruption buffer (1/3, w/v) formed by supplementing the above buffer with 5 mM 2-mercaptoethanol, 0.1 mM Na2EDTA, 1 mM benzamidine and 1 mM of protease in- hibitors [4-(2-aminoethyl)-benzenesulfonyl ﬂuoride hydro- chloride, AEBSF, Pefablock]. The cell extract, obtained by sonication of the cell suspension and centrifugation at 105 000g, was dialyzed against potassium phosphate buffer 20 mM, pH 7.2, containing 0.5 mM Na2EDTA and 5 mM 2-mercaptoethanol, and adsorbed on a DEAE-cellulose column followed by elution with a linear gradient of potassium phosphate from 25 to 250 mM pH 7.2. The AspRS-containing fractions, eluted between 130 and 150 mM salt, were then adsorbed on a Ni-NTA column in 50 mM Tris–HCl buffer pH 7.5 before elution of the proteins with a linear gradient from 25 to 450 mM of imidazole. Pure AspRS (10 mg at 95% purity) were obtained from 30 g cells. The GatCAB containing frac- tions eluted on DEAE-cellulose from 160 to 190 mM po- tassium phosphate were adsorbed on a Ni-NTA column in 50 mM Tris–HCl buffer pH 7.5. After washing with the buffer containing 25 mM imidazole, GatCAB was eluted with 450 mM imidazole. Pure GatCAB (50 mg at 95% purity) was obtained from 80 g of cells. The reconstituted tRNAAsn gene from T. thermophilus was cloned in the pKK223 vector and puriﬁed as described (21). The E. coli (Ec) tRNAAsp gene was ampliﬁed from genomic DNA and cloned into the pUC18 vector for expression in the E. coli JM103 strain. Transfer RNAAsp (800 mg from 120 g of cells; accepting capacity 30.6 nmol.mg 1) was obtained by phenol extractions followed by chromatographies on DEAE-cellulose and nmol.mg Sepharose 4B as described (37). All tRNAAsp and tRNAAsn mutants used in this study were cloned into the pUC18 vector downstream a T7 promoter, transcribed and puriﬁed as described (38).