6092 Nucleic Acids Research, 2006, Vol. 34, No. 21 necessity to use in vitro transcribed tRNAGln and tRNAGlu variants. The crystallographic structure of Staphylococcus aureus GatCAB was published while this manuscript was being completed. This structure and the biochemical results obtained by gel-shift assays of the complexes formed between mutated protein and tRNAGln transcripts have shown that tRNAGln binds to the GatB subunit of the enzyme and that recognition involves the U1–A72 base pair and the D- loop of tRNA (25). Our results are compatible with the pres- ence on the GatCAB enzyme of a unique binding site for Asp-tRNAAsn and Glu-tRNAGln. This conclusion is supported by the 3D structure of S.aureus GatCAB which shows one active site formed by the glutaminase and transamidase cat- alytic sites respectively located in the GatA and GatB sub- units. They are connected by a 30 s-long tunnel which allows channelling of the ammonia from the glutaminase cat- alytic site to the transamidase one (25). Finally, one can wonder how the AdTs are capable to select tRNAAsn and tRNAGln out of 60 species of tRNA pre- sent in the organism, on the basis of a single base pair as posi- tive element for discrimination. We therefore investigated the nature of bp 1–72 in tRNA molecules from the 84 bacterial species using AdT to form Asn-tRNAAsn and/or Gln-tRNAGln and for which the sequences of the full set of tRNA encoding genes are available. The output of this analysis indicates that with the exception of a tRNAThr in Shigella ﬂexneri (26), tRNAAsn and tRNAGln are the only tRNA species beginning with a U1–A72 base pair (27) (data not shown). This supports the fact that speciﬁc selection of the Asp-tRNAAsn and the Glu-tRNAGln amongst the other aa-tRNAs more particularly Asp-tRNAAsp and Glu-tRNAGlu can be achieved on the basis of the recognition of this single base pair. CONCLUDING REMARKS Our investigation shows that the ﬁrst base pair, U1–A72, of bacterial tRNAAsn acceptor arm, constitutes the unique struc- tural element conferring to this tRNA the function of an essential cofactor in Asn biosynthesis (Figure 6A). Although our kinetic study suggests that the mode of recognition of the tRNAAsn moiety of Asp-tRNAAsn by bacterial AdTs might be base-speciﬁc, our results are compatible with another interpretation. Assuming that disruption of the U1–A72 pair of tRNAAsn might be the key event leading to correct presentation of Asp bound to tRNA into the active site before catalysis, the importance of the U1–A72 pair would be related to the weaker hydrogen bonding stability of this base pair compared to the G1–C72 pair of tRNAAsp, leading to a smaller free energy cost of base pair denaturation. In fact, it has been shown that disruption of the U1–A72 base pair of E.coli tRNAGln, induced by binding to GlnRS, allows discrimination of the cognate tRNA from non-cognate tRNAs by the enzyme (28,29). In E.coli GlnRS, a single leucine residue (Leu136) at the tip of a b turn in the acceptor-binding domain stabilizes this disruption through hydrophobic stacking of its side chain with A72 and G2 of tRNAGln. Mutation of either Leu136 or the residues that affect the positioning of its side chain resulted in a relaxed tRNA speciﬁcity of the enzyme (29). The 3D structure of GatCAB shows elements compatible with recog- nition of the U1–A72 base pair of tRNA by a mechanism Figure 6. Localization of the elements determining (A and C) or preventing (B and D) amidation of Asp bound respectively to bacterial (A and B) and archaeal (C and D) tRNAAsn and tRNAAsp. The ribbon or surface representations of the 3D structures of tRNAAsn and tRNAAsp are compared. Since the 3D structures of bacterial and archaeal tRNAAsn and of archaeal tRNAAsp are not available, we used the known 3D structures of tRNA species displaying the bacterial and archaeal elements promoting or preventing transamidation to visualize the tRNAAsn transamidation identity elements and the tRNAAsp transamidation antideterminants. Therefore, the U1–A72 base pair conferring bacterial transamidation identity to tRNAAsn (A) and the V-loop nucleotides G46 D47 of archaeal tRNAAsp (D) are shown in the structural context of yeast tRNAAsp. Similarly, nucleotides G46 and U47 conferring transamidation identity to archaeal tRNAAsn (C) and nucleotide U20A preventing amidation of bacterial tRNAAsp (B) are shown in the structural context of E.coli tRNAAsp. Transamidation identity elements of tRNAAsn (A and C) are represented by yellow spheres or surfaces, and their corresponding counterparts on tRNAAsp (B and D) are highlighted in grey ribbon parts or surfaces. tRNAAsp U20A anti-determinant is represented by a red sphere and surface. resembling that of GlnRS. Indeed, the C-terminal domain of the GatB subunit, which forms the transamidation active site, contains two turn loops and a conserved leucine residue (L472) whose deletion causes loss of tRNA-binding capacity (25). These elements are presumably good candidates for dis- crimination the U1–A72 base pair of tRNAAsn and tRNAGln from G–C pair of tRNAAsp and tRNAGlu. Discrimination between the cognate and non-cognate sub- strates, respectively, Asp-tRNAAsn and Asp-tRNAAsp by bac- terial AdTs does not solely rest on the nature of the ﬁrst base pair of the two tRNAs but also on the length of their D-loop