4970 Nucleic Acids Research, 2012, Vol. 40, No. 11 interpreted through the strong binding of tRNAAsn on GatCAB and the low afﬁnity of the D-AspRS for tRNAAsn. In contrast to the H. pylori system, GatCAB decreases the rate of Asp-tRNAAsn formation by Ec D-AspRS presumably through sequestration of tRNAAsn limiting the free tRNAAsn available for aminoacylation. Thus, the enhancing effect of GatCAB is restricted to tRNAAsn aspartylation by ND-AspRS. In vivo experiments conﬁrmed that expression of Hp ND-AspRS in the transformed E. coli ER Asn- auxotrophic strain was toxic presumably because of the ac- cumulation of Asp-tRNAAsn (Supplementary Figure S2), as previously shown (32). An antibiogram-like test with an Figure 4. Effects of GatCAB on aminoacylation of tRNAAsn using Hp ND-AspRS versus Ec D-AspRS. The graph shows the effect of increasing GatCAB concentrations on initial rates of both AspRSs in the presence of saturating tRNAAsn concentration. Results (v) were normalized using the steady-state rate value of the corresponding AspRS in absence of GatCAB (v0). Concentration of the GatCAB/ tRNAAsn complex increases together with GatCAB concentration. (A) GatCAB increases the steady-state rate of Hp ND-AspRS when tRNAAsn is present (gray triangles), but not when tRNAAsp is used (inset). Error bars represent the SD from three independent experi- ments. (B) GatCAB decreases the steady-state rate of tRNAAsn misacylation with Ec D-AspRS. IPTG-soaked paper disk clearly triggers a growth defect phenotype compared to controls (Supplementary Figure S2A–C). Co-expression of Hp GatCAB reduces this toxic effect, since the enzyme is able to convert Asp-tRNAAsn into Asn-tRNAAsn allowing growth of the recombined strain (Supplementary Figure S2D), although with lesser efﬁciency. GatCAB helps to release Asp-tRNAAsn from the ND-AspRS active site The acceleration of the steady-state rate of tRNAAsn aspartylation by GatCAB (Figure 5) suggests that GatCAB controls the release of Asp-tRNAAsn from the ND-AspRS active site. To test this hypothesis, we used a strategy to nullify this effect without altering GatCAB or ND-AspRS. In other words, we asked whether this effect was solely due to the ability of GatCAB to bind Asp-tRNAAsn. To this end, we investigated aspartylation of tRNAAsn variant mutated at positions known to deter- mine recognition by GatCAB (38). The U1–A72 base-pair constitutes the major transamidation determinant, but recognition of tRNAAsn is further favored by the absence of the U20A position in the D-loop (38). Importantly, these positions are not critical for ND-AspRS recognition (38,42). Thus, we constructed a G1–C72:U20A-containing tRNAAsn variant and veriﬁed that it is a suitable substrate for ND-AspRS but is not recognized by GatCAB (Figure 6 and 38). The aminoacylation kinetics of this tRNAAsn variant remained biphasic and saturating levels of GatCAB had no effect on the steady-state rate of aminoacylation (Figure 6A and B). Thus, removing the ability of GatCAB to bind this mutant Asp-tRNAAsn eliminated the impact of GatCAB on ND-AspRS kinetics. These results indicate that GatCAB enhances ND-AspRS kinetics by recognizing the wild-type aspartylated extrem- ity of tRNAAsn according to the transamidation determin- ants, consequently freeing the ND-AspRS active site. As a Figure 5. GatCAB inﬂuences aminoacylation kinetics for tRNAAsn with Hp ND-AspRS. Pre-steady-state kinetics were conducted under the same conditions of pre-steady-state as previously (4 C, 15% glycerol). (A) In the presence of saturating GatCAB concentration (10 mM), the steady-state rate of tRNAAsn misacylation increases and the burst value slightly decreases. These results suggest that GatCAB increases the dissociation of Asp-tRNAAsn. (B) Zoom on the 4 ﬁrst aminoacylation cycles (in the presence of GatCAB), corresponding to the gray zone in (A). SE was <5% on all values.