Table 1. Kinetic constants for aspartylation of tRNAAsp and tRNAAsn and a tRNA variant with H. pylori ND-AspRS in the absence and presence of GatCAB tRNAAsp tRNAAsn yeast tRNAAsp tRNAAsn (G1–C72, U20A) GatCAB Burst +GatCAB kcat (s 1) KM (mM) 0.33 ± 0.01 n.d. 0.14 ± 0.02 0.08 ± 0.01 + 0.29 ± 0.03 n.d. 0.26 ± 0.02 n.d. + kcat (s 1) KM (mM) 0.31 n.d. 0.25 ± 0.03 0.3 ± 0.03 n.d. n.d. 0.28 ± 0.02 n.d. Burst indicates the presence of an initial fast phase of tRNA aminoacylation. tRNAAsn. The enzyme aspartylates tRNAAsp 2.5-fold faster than tRNAAsn (0.33 and 0.14 s 1), as reported pre- viously (32). However, pre-steady-state kinetics conducted under conditions that allowed an examination of the ﬁrst catalytic cycles of the enzyme (4 C and in the presence of 10 or 15% of glycerol) revealed intriguing differences in aminoacylation of both tRNAs since in contrast to tRNAAsp the charging kinetics of tRNAAsn were biphasic. Aminoacylation of tRNAAsp remains linear after completion of the ﬁrst catalytic cycle. Thus, this ﬁrst cycle occurs with the same rate as subsequent ones (0.041 s 1) (Figure 3). Since ATP-PPi exchange rate is sig- niﬁcantly faster than tRNA charging (15.9 and 0.33 s 1 at 37 C), the steady-state rate of tRNAAsp aminoacylation is dictated by the rate of transfer of activated Asp onto tRNAAsp. Surprisingly, when tRNAAsn is used as a sub- strate, biphasic kinetics arise, which exhibit a burst of Asp-tRNAAsn formation ( 0.04 s 1) followed by a signiﬁ- cantly slower linear phase (0.0033 s 1) (Figure 3). Extrapolation of the linear phase at t0 points to formation of one Asp-tRNAAsn per ND-AspRS active site during the fast phase (Figure 3) and suggests that the ﬁrst tRNAAsn is aspartylated signiﬁcantly faster than those following. Interestingly, the ﬁrst tRNAAsn is aminoacylated with a rate equivalent to that observed for tRNAAsp (Figure 3). Burst and steady-state rate values are both dependent on enzyme concentration (Supplementary Figure S1A). In the slow phase, the steady-state rate of tRNAAsn charging in- creases with the pH but not signiﬁcantly with the ionic strength (Supplementary Figure S1B). This kinetic behavior is consistent with the release of Asp-tRNAAsn being the rate-limiting step at the steady-state of the reaction (40). The slow dissociation of the Asp-tRNAAsn product agrees with the absence of detectable hydrolysis of its ester bond in the presence of ND-AspRS (28). GatCAB modiﬁes the kinetic properties for tRNAAsn Table 1 shows the effect of GatCAB on kinetic constants of Hp ND-AspRS for tRNAAsn and tRNAAsp aspartylation. The addition of GatCAB increases the steady-state rate of Asp-tRNAAsn formation according to a hyperbolic curve (Figure 4A), but has no effect on the steady-state rate of tRNAAsp charging (Figure 4A, Nucleic Acids Research, 2012, Vol. 40, No. 11 4969 Figure 3. Aminoacylation kinetics for tRNAAsp and tRNAAsn with Hp ND-AspRS: a dual response to substrates. Experiments were con- ducted in 15% glycerol at 4 C for tRNAAsp (black diamonds) and tRNAAsn (gray triangles). Results show that tRNAAsp and tRNAAsn are aminoacylated differently. In case of tRNAAsp, the ﬁrst cycle (1 Asp-tRNA formed) has the same rate as the subsequent ones (0.04 s 1). However, in case of tRNAAsn, the ﬁrst cycle (0.04 s 1), which is equivalent to that for tRNAAsp, is signiﬁcantly faster than the subsequent ones (0.0033 s 1). Extrapolation of this slower phase at t0 points to the formation of 1 Asp-tRNAAsn per ND-AspRS active site. This pattern suggests a rate-limiting step which would be the release of Asp-tRNAAsn. SE was <5% on all values. inset and Table 1), indicating that this phenomenon is speciﬁc to tRNAAsn. When saturating concentrations of GatCAB are reached, the rate of tRNAAsn aspartylation increases 1.8-fold (0.14–0.25 s 1) and nearly ﬁts that of tRNAAsp (0.33 s 1, Table 1), while the KM value of ND-AspRS for tRNAAsn increases 3.8-fold, leading to an increase in the overall charging efﬁciency (kcat/KM) of 1.8-fold (Table 1). Thus, in the presence of GatCAB, aminoacylation of tRNAAsn occurs with a similar efﬁ- ciency as that of tRNAAsp (kcat/KM, respectively of 0.27 and 0.30 mM 1 s 1). Pre-steady-state kinetics reveal that this GatCAB-mediated effect originates in an increase in the rate of aminoacylation during the slow phase (0.0033–0.012 s 1, Figure 5A and B). The rate of the fast phase does not seem to be affected (Figure 5B). Considering that tRNAAsn binds GatCAB and ND-AspRS with KD values of 2.1 and 21.4 mM, respect- ively (Figure 1), and that aspartylation of tRNAAsn is more efﬁcient when it is ‘labeled’ with or ‘presented’ by GatCAB (Table 1), the GatCAB/tRNAAsn complex may constitute a better substrate for misacylation by the ND-AspRS. We named this complex the tRNAAsn-pres- entation complex (tRNPC). Since this complex is non-productive in absence of ND-AspRS and can also be a substrate, tRNPC can be considered as a bona ﬁde transfer ribonucleoprotein (tRNP). Interestingly, the tRNPC has opposing effects on the discriminating AspRS of E. coli (D-AspRS) (Figure 4B) in vitro. Ec D-AspRS is able to aspartylate tRNAAsn, in addition to its cognate tRNAAsp, albeit much less efﬁ- ciently (14). As shown for other mischarging kinetics of non-cognate tRNAs, the transfer step is rate-limiting (41). GatCAB decreases the rate of aminoacylation of tRNAAsn by the D-AspRS (Figure 5B). This result can be