6084 Nucleic Acids Research, 2006, Vol. 34, No. 21 by half of the archaeal genomes known so far. However, all archaea use an additional, archaeal-speciﬁc AdT of dimeric structure (GatDE) encoded by the gatD and gatE genes, which only amidates Glu-tRNAGln (7). Despite the predominant role they play in prokaryotic translation, until recently only few structural and functional data were available for these enzymes (2,8). A recent report describing the 3D structure of Pyroccoccus abyssi GatDE AdT partially ﬁlled up the lack of structural information available for this family of enzymes (9). However, the archi- tecture of the active site of these enzymes and their mode of recognition of the aa-tRNA substrates still remain elusive and the structure/function relationship of this family of enzymes is poorly understood. Besides Asn and Gln, two other genetically encoded amino acid, cysteine (Cys) and selenocysteine (Sec) are synthesized via a tRNA-dependent pathway involving modiﬁcation of an amino acid mischarged onto tRNA. Sec-tRNASec, present in many organisms, is formed by modiﬁcation of Ser- tRNASec with selenocysteine synthase (10), whereas in the methanogenic archaea lacking cysteinyl-tRNA synthetase, Cys-tRNACys is formed by modiﬁcation of O-phosphoseryl- tRNACys by O-phosphoseryl-tRNA:Cys-tRNA synthase (11). It is still unknown how these enzymes bind the mischarged aa-tRNA molecule and, more important, what are the struc- tural elements of the tRNA molecule that ensure accurate recognition and reliable enzymatic transformation of the mischarged amino acid. We focused our interest on recognition of tRNA by the bacterial GatCAB AdT because of the peculiar properties of this enzyme among the AdT family. In contrast to GatDE, GatCAB AdT is able to transform two different amino acids, yielding two different cognate aa-tRNA pairs. In order to efﬁciently achieve that goal, the enzyme has to be speciﬁc enough in order to discriminate the incorrectly charged Asp-tRNAAsn from cognate Asp-tRNAAsp and the micharged Glu-tRNAGln from cognate Glu-tRNAGlu. The dis- crimination capacity of this enzyme is of extreme importance since amidation of Asp-tRNAAsp into Asn-tRNAAsp or of Glu-tRNAGlu into Gln-tRNAGlu would form aa-tRNA spe- cies, that, unlike Asp-tRNAAsn or Glu-tRNAGln, have been predicted to be efﬁcient EF-Tu binders and therefore would lead to misincorporation of amino acids into proteins (12,13). However, speciﬁcity of AdTs should also be relaxed enough to form two correctly paired aa-tRNAs, Asn-tRNAAsn and Gln-tRNAGln. The mechanism of aa-tRNA discrim- ination by AdT has not been explored so far and it is not known whether identical tRNA elements trigger recognition of both Asp-tRNAAsn and Glu-tRNAGln or if the two tRNA moieties are recognized through distinct elements. In addition, despite the high degree of sequence similarity between the archaeal and bacterial GatCAB AdTs, the possibility that both enzymes utilize different modes of tRNA recognition cannot be excluded. It would therefore be interesting to verify whether AdTs, like aaRSs, exhibit kingdom-speciﬁc tRNA recognition patterns. To gain insight on how Asp-tRNAAsn and Glu-ARNtGln are recognized and are discriminated from correctly charged Asp-tRNAAsp and Glu-ARNtGlu by GatCAB AdTs we inves- tigated the structural elements distinguishing tRNAAsn from tRNAAsp and tRNAGln from tRNAGlu and which should be the potential determinants of speciﬁc amidation of tRNA- bound amino acid. We started our investigation by searching the structural elements of tRNAAsn that promote conversion of the bound Asp into Asn by the bacterial GatCAB, and those of tRNAAsp preventing this conversion. We chose for this study the partners of the tRNA-dependent pathway of Asn formation from Neisseria meningitidis, a human patho- genic bacterium, deprived of AsnRS and thus using the AdT pathway to form Asn-tRNAAsn (14). We show that a sin- gle base pair, U1–A72 determines amidation of Asp bound to tRNAAsn by bacterial AdT whereas the supernumerary U20A in the D-loop prevents amidation of Asp bound to tRNAAsp. We extended the investigation to the archaeal system and showed that tRNA-dependent Asn formation is promoted by the length of the variable region. Cross-species trans- amidation experiments together with sequence-based com- parison of archaeal and bacterial tRNAAsp and tRNAAsn, indicate that transamidations by bacterial and archaeal GatCAB AdTs involve kingdom-speciﬁc tRNA elements. Finally, on the basis of the analysis of the sequences of tRNAGln and tRNAGlu from bacteria using AdT to form Gln-tRNAGln, we predict that bacterial AdTs use the same tRNA structural elements to catalyze the tRNA-dependent formation of Gln and Asn. MATERIALS AND METHODS Materials TLC cellulose plates (20 · 20 cm2) were from Merck, hydro- xyapatite CHT20 and UNO-Q6 columns from BIO-RAD, DEAE–cellulose DE-52 from Whatman and Heparine- Ultrogel and Cibacron blue-Sepharose from Pharmacia. L-[14C]Asp (207 mCi/mmol) was from Amersham, Dynazyme Taq polymerase from Finnzyme, restriction enzymes and the pCYB1 vector were from NEW ENGLAND Biolabs. Epicu- rian Coli BL21-CodonPlus -RIL competent cells were from Stratagene and N.meningitidis C58 genomic DNA from ATCC. AdT from N.meningitidis, Thermus thermophilus and Methanosarcina barkeri The AdT from N.meningitidis was expressed in the Escherichia coli BL21 CodonPlus RIL strain transformed with the pCYB1 vector recombined with the reconstituted, artiﬁcial, gatCAB operon. The protein was puriﬁed from the S100 extract by chromatographies on DEAE–cellulose, Phosphocellulose, Hydroxyapatite, Heparine-Ultrogel, Uno-Q6 and Cibacron blue-Sepharose. Pure enzyme (180 mg) was obtained from 35 g of cells. The pure enzyme was free of traces of E.coli asparagine synthetases A and B, since incubation of 10 mM of the protein in a standard amidation mixture containing 50 mM of [14C]Asp did not lead to formation of a detectable amount of [14C]Asn (data not shown). The AdT from T.thermophilus was puriﬁed as described previously (15). AdT-enriched protein fractions from M.barkeri were obtained by chromatography of an S100 extract on DEAE–cellulose and identiﬁed by western blot with antibodies directed against T.thermophilus AdT. The dialyzed fractions were stored at 20 C in 100 mM Na–HEPES buffer, pH 7.2, containing 5 mM