Table 1. Amidation kinetic constants of aspartylated wild-type and mutated tRNAAsn and tRNAAsp transcripts by N.meningitidis AdT Asp-tRNA transcripts Asp-tRNAX Asn wild-type transcript Asn U1–G72 Asn C36 Asn + U20A Asn G1–C72 Asn G1–C72,+ U20A Asn G1–C72, + U20A, C36 Asp wild-type Asp U1–G72 Asp DU20A Asp U1–G72, DU20A Asp U1–A72 Asp U1–A72, DU20A Asp U1–A72, DU20A, U36 Kinetic constants KM (mM) 1.2 5.6 0.48 11.93 n.d. n.a. n.a. n.a. n.a. n.a. n.d. n.d. 2.7 0.6 LKM 1.0 5.0 0.4 10.0 n.d. n.a. n.a. n.a. n.a. n.a. n.d. n.d. 2.0 0.5 Nucleic Acids Research, 2006, Vol. 34, No. 21 6087 kcat (s 1) 0.64 0.46 0.65 0.91 n.d. n.a. n.a. n.a. n.a. n.a. n.d. n.d. 0.68 0.46 Lkcat 1.0 1.4 1.0 0.7 n.d. n.a. n.a. n.a. n.a. n.a. n.d. n.d. 0.9 1.4 kcat/KM (mM 1 s 1) 0.53 0.082 1.35 0.076 0.0052 n.a. n.a. n.a. n.a. n.a. 0.0022 0.0044 0.25 0.77 Lkcat/KM 1.0 6.4 0.4 7.0 102.0 n.a. n.a. n.a. n.a. n.a. 241.0 120.0 2.0 0.7 The tRNA species (X ¼ Asn or Asp) and the mutated nucleotides are indicated. D and + indicate the deletion of a nucleotide and the introduction of a supernumerary nucleotide, respectively. Each KM and kcat value represents the average of at least two independent measurements and their standard deviations were evaluated at 20%; n.a.: not amidated, n.d.: not determined. The losses of affinity (quantified by the increase of KM, LKM), of the catalytic rate constant (Lkcat) and of the catalytic efficiencies (Lkcat/KM) induced by the mutations are expressed, respectively, by the following ratios: KM of the Asp-tRNA variant over KM of wild-type Asp-ARNtAsn, kcat of wild-type Asp-ARNtAsn over kcat of the Asp-tRNA variant and kcat/KM of wild-type Asp-ARNtAsn over kcat/KM of the Asp-tRNA variant. always 1 nt shorter than in tRNAAsp because of the absence of base 20A, which, in tRNAAsp, is mainly a U (Figure 2A). Among these candidates, we deﬁned as transamidation identity elements the nucleotides from tRNAAsn that upon swapping with their tRNAAsp counterparts would signiﬁ- cantly decrease the amidation efﬁciency of the Asp bound to the tRNAAsn variant, whereas transplantation of these elements of tRNAAsn in tRNAAsp should trigger amidation of Asp attached to the tRNAAsp variant. To validate our approach we ﬁrst generated a tRNAAsn and a tRNAAsp variant in which all the elements we identiﬁed were exchanged by their tRNAAsp and tRNAAsn counterparts, respectively. We thus synthesized the tRNAAsnðG1-C72‚ þU20A‚ C36Þ mutant, in which the U1–A72 base pair was mutated into a G1–C72 pair, U36 was replaced by C36 and the D-loop was extended by introduction of a U in position 20A. In the tRNAAspðU1-A72‚ DU20A‚ U36Þ mutant, the G1–C72 base pair was mutated into a U1–A72 pair, C36 was replaced by U36 and the D-loop was shortened by deletion of position U20A. The two variants were able to be amino- acylated with Asp and they could subsequently be subjected to a transamidation reaction in conditions allowing KM and kcat measurements (Materials and Methods). The kinetic analysis shows that Asp-tRNAAsnðG1-C72‚ þU20A‚ C36Þ loses com- pletely its capacity to be amidated by N.meningitidis AdT whereas Asp-tRNAAspðU1-A72‚ DU20A‚ U36Þ acquires the capability of being amidated as efﬁciently as the wild-type Asp- tRNAAsn (Figure 3A and Table 1). This result validates the alignment strategy we used to identify the complete set of the potential tRNA transamidation identity elements. To analyze the individual contribution of each element and to characterize the minimal set of elements promoting trans- amidation we generated T7 transcripts of N.meningitidis tRNAAsn variants where the U1–A72 base pair and U36 were individually exchanged by their corresponding counterparts of N.meningitidis tRNAAsp (tRNAAsnðG1-C72Þ and tRNAAsnðC36Þ). To test the inﬂuence of the length of D-loop in transamidation of Asp-tRNAAsn we also created a tRNAAsn mutant in which U20A was introduced in the D-loop (tRNAAsnðþU20AÞ) and a tRNAAsp mutant in which U20A was deleted (tRNAAsnðDU20AÞ). Figure 3A and Table 1 show that tRNAAsnðC36Þ mutant displaying the tRNAAsp anticodon pro- motes amidation of the bound Asp only two times less efﬁ- ciently than the wild-type tRNAAsn. This result clearly indicates that the anticodon of the tRNAs we tested does not contribute to the transamidation catalyzed by the GatCAB AdT. Introduction of U20A in the D-loop of tRNAAsn resulted in a loss of transamidation efﬁciency by almost one order of magnitude as a consequence of a decrease in afﬁnity of the Asp-tRNAAsnðþU20AÞ variant for AdT. Deletion of this nucleotide in the D-loop of tRNAAsp is, however, not sufﬁ- cient to confer capacity to tRNAAsp to trigger amidation of the bound Asp. Mutation of the ﬁrst base pair of the acceptor arm of tRNAAsn creates the tRNAAsnðG1-C72Þ variant whose aspartylated form is still amidated (Figure 3A) but 100-fold less efﬁciently than Asp-tRNAAsn (Table 1). This loss is mainly attributed to a severely decreased afﬁnity of the aspartylated tRNAAsn variant for AdT. As a consequence, individual kinetic constants could not be measured for this variant, and therefore only the catalytic efﬁciency was determined (19). The above-mentioned results strongly suggest that the ﬁrst base pair of the acceptor arm of tRNAAsn is a major element determining tRNA identity for transamidation by AdT. Indeed, transplantation of this element into tRNAAsp generates the tRNAAspðU1-A72Þ mutant whose aspartylated form is amidated. However, Asp-tRNAAspðU1-A72Þ still exhibits a loss in catalytic efﬁciency, of two orders of magnitude, compared to the wild-type Asp-tRNAAsn and Asp-tRNAAspðU1-A72‚ DU20A‚ U36Þ. The difference in catalytic efﬁciencies observed between Asp-tRNAAspðU1-A72Þ and Asp-tRNAAspðU1-A72‚ DU20A‚ U36Þ can be attributed to different nucleotides in position 36 or to distinct sizes of the D-loops of the two tRNAs. However, we showed that position 36 of the anticodon does not belong to the identity elements. Fur- ther, nucleotide U20A being absent in the D-loop of tRNAAsn