6090 Nucleic Acids Research, 2006, Vol. 34, No. 21 To establish that nucleotides G46 and U47 of the V-region of tRNAAsn are involved in the archaeal transamidation, we performed cross-species reactions using archaeal AdT and transcripts of yeast tRNAAsp and of the yeast tRNAPhe variant (tRNAPhe!Asp) containing the aspartate aminoacylation identity elements, both efﬁciently aspartylated by yeast AspRS (16). Both tRNAs contain the U12–A23 base pair in the D-stem, but while yeast tRNAAsp displays the V-region of archaeal tRNAAsp (absence of position 47 and A46), the tRNAPhe!Asp V-region resembles that of archaeal tRNAAsn (G46 U47, Figure 4). As a consequence, these tRNAs are suitable substrates for analyzing the role of the V-region in transamidation by the archaeal AdT. Figure 4D and E shows that the archaeal AdT amidates Asp bound to yeast tRNAPhe!Asp but not when bound to yeast tRNAAsp, conﬁrm- ing that transamidation by archaeal AdT is promoted by nucleotides G46 and U47 of the V-region. This agrees with the incapacity of archaeal AdT to amidate Asp bound to yeast tRNAAsp containing the U12–A23 pair. Thus this base pair, like U36, does not constitute a determinant for archaeal transamidation. However, these results do not allow to conclude whether the identity is determined by the length of the V-region or by the nature of nt 46 and 47; nor they allow to conclude whether these positions are identity elements per se or not, because of the critical role they play in the folding of tRNA and in the stability of the L-shaped structure. Nt 46 interacts via three H-bonds with the 13–22 bp of the D-stem, and it has been shown that lack of nt 47 alters the stability of tRNA (22). Therefore discrimination by archaeal AdT between Asp-tRNAAsn and Asp-tRNAAsp could be based on the distinct 3D structures that both tRNAs adopt. Com- parison of the tRNA sequences of archaea that use the tRNA-dependent pathway to form Asn shows that, in each species, 9–25 tRNAs contain G46 and U47 in the V-region. Therefore, selection of Asp-tRNAAsn by the archaeal AdT is likely achieved by the Asp moiety. These observations sug- gest that the Asp moiety contributes more to recognition of Asp-tRNAAsn by archaeal AdT than by the bacterial homolog where tRNAAsn and tRNAGln are the unique tRNAs, among all species, to display the elements determining amidation. Predicting what are the identity elements for amidation of Glu bound to tRNAGln Independently of the activity they carry out in vivo (Asp- or/ and Glu-AdT), all bacterial AdTs studied so far exhibit in vitro dual speciﬁcity and form both Asn-tRNAAsn and Gln-tRNAGln. One of the questions raised by the functional property of AdT is whether tRNAAsn and tRNAGln display the same set of recognition elements for amidation of the bound amino acids or if AdTs recognize distinct elements of the two tRNA species. To gain insight into the elements of tRNAGln promoting amidation of the bound Glu and those of tRNAGlu preventing the amidation we aligned several tRNAGln and tRNAGlu sequences from bacteria using the transamidation pathway to form Gln-tRNAGln. The comparative alignment shows that the elements conserved in tRNAGln but which differ in tRNAGlu are those, which in tRNAAsn, promote conversion of bound Asp into Asn, and those, which in tRNAAsp, prevent Figure 5. Comparative alignment of tRNAGln and tRNAGlu sequences from bacteria using AdT to form Gln-tRNAGln. The cloverleaf structures of the consensus sequences of 101 bacterial tRNAGlu and tRNAGln 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. Blue nucleotides are conserved in tRNAGlu and tRNAGln. White nucleotides in red boxes correspond to nucleotides strictly conserved in all tRNAGln species but which differ in all tRNAGlu species. White triangle in red boxes correspond to the lack of a given nucleotide in all tRNGln species as compared to the tRNAGlu species. this conversion (Figure 5). This set of elements is constituted by the ﬁrst base pair of the tRNA acceptor arm, a U1–A72 pair present in tRNAGln and in tRNAAsn which is substituted in tRNAGlu, like as in tRNAAsp by the G1–C72 pair. In addition like tRNAAsp when compared to tRNAAsn, tRNAGlu displays a supernumerary nucleotide in positions 20A or 20B of the D-loop when compared to tRNAGln (Figure 5). Similar to tRNAAsn and tRNAAsp, both tRNAGln and tRNAGlu differ by the 30 nucleotide of the anticodon, G36 in tRNAGln and C36 in tRNAGlu. However, since this nucleotide does not sig- niﬁcantly contribute to amidation of Asp bound to tRNAAsn, we can predict that AdTs do not discriminate tRNAGln from tRNAGlu on the basis of this nucleotide. These results suggest that the same structural elements of AdT are involved in selection of both Asp-tRNAAsn and Glu-tRNAGln. Further support of this prediction is brought by the following facts: (i) in bacteria that use both Asp-AdT and Glu-AdT activities, tRNAAsn and tRNAGln contain a U1–A72 base pair while tRNAAsp and tRNAGlu contain a G1–C72 pair and an extended D-loop (Table 2); (ii) in bacteria utilizing only the Asp- or the Glu-AdT activity, the tRNA that promotes the amidation of the bound amino acids, always contains the elements conferring transamidation identity (Table 2); (iii) in bacteria deprived of the AdT, the tRNAAsn and tRNAGln are directly charged by the cognate aaRS and do not contain these elements. Conﬁrming experimentally our prediction of the bacterial tRNAGln transamidation identity set will be difﬁcult to per- form since unmodiﬁed tRNAGlu, derived by in vitro transcrip- tion, have been shown to be poor substrates for all bacterial GluRSs studied so far whether they are discriminating or not. In the case of E.coli GluRS, it has been shown that the cognate tRNAGlu T7 transcript exhibits a 100-fold reduction in its catalytic efﬁciency compared to that of tRNAGlu pre- pared from an overproducing strain (23,24). Thus, conﬁrma- tion of the predicted bacterial tRNAGln identity set cannot be conducted with the kinetic approach which rests on the