6088 Nucleic Acids Research, 2006, Vol. 34, No. 21 Figure 2. Comparative alignment of tRNAAsn and tRNAAsp sequences from bacteria (A) and archaea (B) using AdT to form Asn-tRNAAsn. The cloverleaf structures of the consensus sequences of 49 bacterial (A) and 16 archaeal (B) tRNAAsp and tRNAAsn sequences are compared. Grey circles represent nucleotides conserved in all tRNA species. Purple open circles correspond to positions for which the nature of the base varies in the considered tRNA species. Grey nucleotides are conserved in tRNAAsp and tRNAAsn. White nucleotides in red boxes correspond to positions for which the nature of the base is strictly conserved in all tRNAAsn species but differs in all tRNAAsp species. The white triangle in a red box corresponds to the lack of the considered nucleotide in all tRNAAsn species as compared to the tRNAAsp species. it cannot be considered per se as an identity element, and should therefore more likely act as an anti-determinant that decreases recognition of aspartylated tRNAAsp. To verify this hypothesis we deleted nucleotide U20A from tRNAAspðU1-A72Þ and added a U20A in tRNAAsnðG1-C72Þ. As expected, Asp-tRNAAspðU1-A72‚ DU20AÞ is transamidated nearly as efﬁciently as wild-type Asp-tRNAAsn by N.meningitidis AdT whereas Asp-tRNAAsnðG1-C72‚ þU20AÞ has completely lost its capacity to be amidated (Figure 3A and Table 1). These results clearly show that efﬁcient transamidation of N.meningitidis Asp-tRNAs by the cognate AdT is promoted by a single identity element which is the ﬁrst base pair of tRNAAsn acceptor arm. Moreover, it shows that amidation of Asp-tRNAAsp is prevented by the presence of the super- numerary nucleotide U20A in the D-loop of tRNAAsp which acts as an anti-determinant. By preventing amidation of Asp bound to tRNAAsp, this position improves speciﬁc amidation of Asp bound to tRNAAsn. To verify if the nucleotides in tRNAAsn determining ami- dation of the bound Asp in N.meningitidis, are conserved Figure 3. Analysis of the amidation efficiencies of Asp bound to wild-type and mutated tRNAAsn and tRNAAsp by N.meningitidis (A) and T.thermophilus (B) AdTs. The reactions were conducted and the samples were treated as described in Materials and Methods. The remaining Asp and the Asn formed after 20 min incubation were analyzed in 10 ml aliquots by TLC after deacylation of the [14C]aa-tRNA. The mutations introduced in the tRNAs are indicated on the top of each lane; D and + indicate deletion and insertion of a nucleotide, respectively. in other bacterial species forming Asn by the tRNA- dependent pathway, we analyzed the amidation proﬁle of Asp bound to the various N.meningitidis tRNAAsn and tRNAAsp variants by AdT from a species phylogenetically distant from N.meningitidis, like T.thermophilus. Figure 3B shows that amidation by T.thermophilus AdT requires the same elements of tRNAAsn as the enzyme from N.meningitidis. The minor differences observed concern the relative contribution of each element to the amidation efﬁciency. For the T.thermophilus AdT the importance of A72 seems increased since Asp-tRNAAsnðU1-G72Þ is less well amidated by the T.thermophilus enzyme than by the N.meningitidis one. Additionally, the supernumerary U20A of tRNAAsp is a stronger anti-determinant when amidation is catalyzed by T.thermophilus AdT than by the N.meningitidis enzyme. These conclusions are supported by the following observations: (i) Asp-tRNAAsn(+U20A) is less well ami- dated by AdT from T.thermophilus than by that from N.meningitidis, (ii) Asp-tRNAAsnðD20AÞ is slightly amidated by T.thermophilus AdT but not by that of N.meningitidis and (iii) when deprived of U20A, amidation efﬁciency of Asp-tRNAAspðU1-A72Þ increases more with T.thermophilus AdT than with the N.meningitidis one (Figure 3A and B). However, since the same positions of tRNAAsn and of tRNAAsp, respectively, promote and prevent transamidation by the AdTs from T.thermophilus and from N.meningitidis both enzymes discriminate Asp-tRNAAsn from Asp-tRNAAsp by similar mechanisms. So far among aa-tRNA forming enzymes, only alanyl-tRNA synthetase has been shown to