Nucleic Acids Research, 2012, Vol. 40, No. 11 4971 Figure 6. GatCAB pulls Asp-tRNAAsn from the ND-AspRS active site. (A) Pre-steady-state kinetics performed with a tRNAAsn mutated at the GatCAB identity elements: G1–C72:U20A replace the natural U1–A72: 20A positions. Addition of GatCAB (black diamonds, +GatCAB) has no effect on the steady-state rate, compared to a reaction without GatCAB (gray triangles, GatCAB). (B) Pre-steady-state kinetics performed with a wild-type tRNAAsn. Addition of GatCAB (black diamonds, +GatCAB) accelerates the steady-state rate, compared to a reaction without GatCAB (gray triangles, GatCAB). consequence, the overall turnover of ND-AspRS increases in the presence of GatCAB. Origin of the rate-limiting step What are the structural elements that trigger different aminoacylation mechanisms with respect to tRNAAsp and tRNAAsn? The major differences between bacterial tRNAAsp and tRNAAsn lie within the acceptor arm (dis- playing a G1–C72 or a U1–A72 base pair, respectively) and the anticodon loop (positions C36 and U36 and C38 and A38 in tRNAAsp and tRNAAsn, respectively). Yeast cyto- plasmic tRNAAsp provides a natural variant that displays a GUC ‘Asp’ anticodon and a bacterial ‘tRNAAsn-like’ acceptor-end that almost avoids aminoacylation by Ec D-AspRS (43). Similar to bacterial tRNAAsp, aminoacylation of yeast tRNAAsp is monophasic (data not shown). Thus, the elements leading to the biphasic kinetics for aminoacylation of tRNAAsn by the Hp ND-AspRS are absent in yeast tRNAAsp. Further, substi- tution of C36 by U36 in bacterial tRNAAsp leading to a GUU ‘Asn’ anticodon did not trigger a biphasic kinetic response (data not shown). Hence, the divergent kinetic responses of Hp ND-AspRS for tRNAAsp versus tRNAAsn aminoacylation are probably triggered by different overall conformations of the aminoacyl-tRNA end-products, rather than by distinct afﬁnities for the tRNA substrates. This hypothesis is in line with the fact that the two kcat’s of aspartylation (Table 1) show opposing trends with respect to the afﬁnities of the enzyme for the two tRNAs (Figure 1). Moreover, the steady-state rate of tRNAAsn aspartylation is more affected by increase of the pH than by that of the ionic strength (Supplementary Figure S1), suggesting that the aminoacylated tRNAAsn extremity, and mostly the Asp moiety, plays a prevalent role in the slow dissociation of Asp-tRNAAsn, in accordance with other studies on the rate-limiting step of tRNA aminoacylation (40) and the inhibition properties of Asp-ol-AMP. Interestingly, this non- hydrolyzable Asp-AMP mimic, competitive with respect to Asp and ATP, shows different inhibition patterns for aminoacylation of tRNAAsp and tRNAAsn (39 and Supplementary Table S1). Indeed Ki values of 8 and 32 mM were determined in presence of tRNAAsp and tRNAAsn, respectively (Supplementary Table S1). This 4-fold increase in the Ki value when Asp-tRNAAsn is formed suggests a strong binding of the tRNAAsn-bound Asp moiety within ND-AspRS active site, compared to Asp-tRNAAsp. This is further conﬁrmed with differential inhibition patterns using Asp-ol-AMP (Supplementary Table S1) in presence of tRNAAsp or tRNAAsn, the latter tRNA protecting the active site from inhibition more efﬁciently as a consequence of its retention. This is in agreement with a previous work on Pseudomonas aeruginosa ND-AspRS (39) showing that the limiting step for Asp-tRNAAsn synthesis could be general for ND-AspRS of bacterial type. To obtain further insights into the origin of the distinct dissociation rates of the two aminoacyl-tRNAs from ND-AspRS, we performed Arrhenius activation energy (EA) measurements (Supplementary Figure S3). The EA values are the same for aminoacylation of tRNAAsp (68.6 ± 0.6 kJmol 1K 1) and tRNAAsn (66.9 ± 1.9 kJmol 1K 1). Thus, both EA are determined by the same step, presumably the transfer step, since the release of aminoacyl-tRNA does not contribute signiﬁ- cantly to EA. From these similar EA values, one can suggest that the steps preceding the release of Asp-tRNA are equivalent and that the differences occur after the transfer step. It is therefore reasonable to assume that the formation of Asp-tRNAAsp, or Asp-tRNAAsn trigger different structural arrangements of the active site. This conformational difference may explain the dif- ference in Asp-tRNA release and in inhibition patterns by Asp-ol AMP. DISCUSSION The Asn-transamidosome in H. pylori Previous work has suggested that an Asn-transamidosome should exist in H. pylori (28). Indeed, this organism does