Gel-ﬁltration Experiments were conducted as described (21) using an A¨ KTA Puriﬁer and a 24-ml Superdex G200 column (GE Healthcare) equilibrated with 50 mM Na–HEPES buffer pH 7.2 containing 30 mM KCl, 6 mM MgCl2, 0.1 mM Na2EDTA and 5 mM 2-mercaptoethanol at 12 C. Samples of 0.2 ml containing the enzyme and tRNA partners at the indicated concentrations were diluted in the equilibration buffer and analyzed. KD values [free en- zyme] [free tRNAAsn]/[enzyme/tRNAAsn complex] were determined by evaluation of the areas of enzyme-bound tRNA and free tRNA peaks, the sum of which equals the total tRNA present in the sample. The contaminating non-speciﬁc tRNA was subtracted from free tRNA. The quantity of free enzyme was determined by subtracting the quantity of enzyme-bound tRNA from total amount of enzyme in the sample. Dynamic light scattering Dynamic light scattering (DLS) experiments were con- ducted as described (21) at 20 ± 0.1 C in a Zetasizer NanoS instrument (Malvern, UK). The 20- to 40-ml samples contained 10 mM ND-AspRS (20 mM subunits) and/or 20 mM GatCAB with or without 20 mM tRNAAsn in the gel-ﬁltration buffer. When present, ATP, Asp and Gln were at a concentration of 2 mM. Hydrodynamic diameters were corrected for solvent refractive index (n = 1.3353) and absolute viscosity (Z = 1.041 mPa.s). Aminoacylation of tRNA The reaction mixture contained 100 mM Na–HEPES buffer pH 7.2, 30 mM KCl, 12 mM MgCl2, 2 mM ATP, 20–50 mM [14C] L-Asp (330 cpm.pmol 1, GE Healthcare), 0.1 mg.ml 1 bovine serum albumin, 10 mM T. thermophilus (Tt) tRNAAsn or Ec tRNAAsp, or 0.04–6 mM Tt tRNAAsn for KM measurements, and 0.01–0.5 mM Hp ND-AspRS. The reactions were conducted at 37 C unless otherwise indicated. The rate-limiting step was determined with 0.5, 1.0 and 1.5 mM Hp ND-AspRS and 10 mM tRNAAsn or tRNAAsp without or with GatCAB at the indicated concentrations in the presence of 15% glycerol at 4 C. For KM measurements of Asp and ATP in the presence of tRNAAsp or tRNAAsn, the small substrate concentra- tions varied from 10 to 300 mM and tRNA was present at a 10 mM concentration. Inhibition experiments with aspartol-adenylate (Asp-ol-AMP), kindly provided by Prof. R. Cheˆ nevert (Universite´ Laval, Que´ bec), were per- formed with concentrations ranging from 10 to 200 mM. The inhibition constants were determined as described (39). The [14C] aminoacyl-tRNA formed in 10 or 20 ml aliquots withdrawn at various time intervals was determined as described (1). ATP-PPi exchange The reaction mixture contained 100 mM Na–HEPES buffer pH 7.2, 10 mM MgCl2, 2 mM L-Asp, 2 mM ATP, 2 mM [32P] PPi (2 cpm.pmol 1, Perkin Elmer) and 0.5 mM ND-AspRS. The [32P] ATP formed at 37 C was Nucleic Acids Research, 2012, Vol. 40, No. 11 4967 determined in 40 ml aliquots at various time intervals as described (1). Transamidation of Asp-tRNAAsn Reactions were conducted at 37 C for 10 min in a mixture containing 100 mM Na–HEPES buffer pH 7.2, 30 mM KCl, 12 mM MgCl2, 2 mM ATP, 10 mM [14C] L-Asp (330 cpm.pmol 1), 1 mM L-Gln, 0.1 mg.ml 1 bovine serum albumin and 10 ml of the transamidosome fraction eluted from size-exclusion chromatography. Transa- midation was measured as described (21,24). RESULTS An Asn-transamidosome in H. pylori Association of ND-AspRS, tRNAAsn and GatCAB from T. thermophilus leads to a stable ternary complex named Asn-transamidosome (21). Gel-ﬁltration and DLS experi- ments conducted with the H. pylori partners show that the ND-AspRS can bind both tRNAAsp and tRNAAsn but with different afﬁnities (KD values of 7.9 and 21.4 mM re- spectively, Figure 1A) and that GatCAB can form a much more stable binary complex with tRNAAsn than ND-AspRS (KD value of 2.1 mM, Figure 1B). These diver- gent KD values differ from those obtained using the T. thermophilus partners, where tRNAAsn bound to ND-AspRS with higher afﬁnity than to GatCAB (21). The asymmetric KD values determined with the H. pylori system were conﬁrmed by DLS (Figure 2). Indeed, a higher-sized particle was clearly seen in the presence of ND-AspRS and tRNAAsp (12.6 nm), compared to free ND-AspRS and tRNA (10.9 and 4.9 nm, respectively) but not when ND-AspRS and tRNAAsn were mixed (Figure 2, lanes 1–4) (10.9 nm). Similarly, we detected an association between GatCAB and tRNAAsn (10.9 nm) compared to their isolated counterparts (9.4 and 4.9 nm, respectively) (Figure 2, lanes 5 and 6). No association of the protein partners in the absence of tRNA was detected by either technique (data not shown). Finally, when the three partners were mixed, a new ribonucleoprotein (RNP) of signiﬁcantly higher size (13.5 nm) appeared (Figure 2, lane 7). Isolation of this complex by gel-ﬁltration (Figure 1C) and analysis of its components by SDS–PAGE (Figure 1D) revealed the presence of ND-AspRS, the three GatCAB subunits and tRNAAsn. Functional analysis conﬁrmed that it was fully able to synthesize Asn-tRNAAsn in the presence of free Asp, ATP and Gln (Figure 1E). Because the association of the protein partners is tRNAAsn-dependent, this complex constitutes a bona ﬁde Asn-transamidosome according to previous arguments (21). Dynamics of the H. pylori Asn-transamidosome In T. thermophilus, the Asn-transamidosome is charac- terized by its remarkable stability in gel ﬁltration (21,22), whereas for H. pylori, it is less stable (Figure 1C). Since this observation could reﬂect a difference in dynamic properties, we tested the stability of the Hp Asn-transamidosome by measuring its hydrodynamic