Nucleic Acids Research, 2006, Vol. 34, No. 21 6089 Figure 4. (A–E) Identification of the archaeal tRNAAsn transamidation identity elements by cross-species transamidation experiments. The transamidation mixture containing 2 mM aspartylated N.meningitidis tRNAAsp or tRNAAsn or aspartylated yeast tRNAAsp or tRNAPhe!Asp or aspartylated tRNAAsn in unfractionated M.barkeri tRNA and either 25 nM N.meningitidis AdT (Ba.) or 5 ml of M.barkeri AdT-enriched fraction (Ar.) was incubated during 60 min at 37 C. The remaining Asp and the Asn formed are determined after deacylation of the [14C]aa-tRNA by TLC analysis as described in Materials and Methods. The inset shows the spots of [14C]Asp and [14C]Asn after scanning of the image plate. The cloverleaf structures of the tRNAs used in these experiments are displayed. In each tRNA species the nucleotides belonging to the archaeal set of transamidation identity determinants predicted by comparative sequence alignment (Figure 2B) are in grey boxes. display a tRNA identity set constituted only by a single base pair (20). Finally we wondered whether the tRNAAsn elements we identiﬁed as being responsible for amidation of the bound Asp, could be brought down to the single U1 base. For this reason, we constructed the tRNAAsnðU1-G72Þ and tRNAAspðU1-G72‚ DU20AÞ variants and analyzed their ability to trigger amidation of the bound Asp by AdT. Figure 3 and Table 1 show that Asp-tRNAAsnðU1-G72Þ is trans- amidated only 6.4 times less efﬁciently than the wild-type Asp-tRNAAsn, but 16 times more efﬁciently than Asp-tRNAAsnðG1-C72Þ. This result suggests that U1 from accep- tor arm is the major identity determinant for amidation and that A72 might be dispensable. Similarly, introduction of U1–G72 in tRNAAspðDU20AÞ confers competency to tRNAAsp to promote amidation of the acylating Asp by AdT. However, the tRNAAspðU1-G72;DU20AÞ variant promotes amida- tion of the bound Asp 120-fold less efﬁciently than the tRNAAspðU1-A72‚ DU20AÞ variant. This indicates that nucleotide A72 contributes more efﬁciently to amidation of Asp by AdT in the framework of tRNAAsp than in that of tRNAAsn. Kingdom-specific tRNA elements for tRNA-dependent asparagine formation Besides bacteria, almost half of the archaea use also a GatCAB AdT to form Asn-tRNAAsn (7,21). The archaeal GatC, GatA and GatB subunits share a high degree of sequence homology with their bacterial counterparts and both AdTs display the same functional properties since they transamidate in vitro both Asp-tRNAAsn and Glu-tRNAGln (7). However, the tRNA elements promoting transamidation differ for the two phylae. In archaea, the ﬁrst base pair of tRNAAsn, a G1–C72 pair, is also present in tRNAAsp (Figure 2B), indicating that, in contrast to bacteria, this base pair cannot speciﬁcally promote transamidation of Asp-tRNAAsn. Applying the strategy of comparative tRNA alignments to the archaeal tRNAAsp and tRNAAsn species, we identiﬁed the U12–A23 base pair from the D-stem, nucleotide U36 and nucleotides G46 and U47 from the V-region of tRNAAsn as the potential identity elements promoting tRNA-dependent transamidation by the archaeal AdT (Figure 2B). These nucleotides differ in archaeal tRNAAsp where the 12–23 bp is mainly G–C, C is found in position 36 and the V-region is 1 nt shorter than in tRNAAsn because nt 47 is missing whereas A is found in position 46 (Figure 2B). Interestingly, the elements that could determine amidation of Asp bound to archaeal tRNAAsn are conserved in all bacterial tRNAAsn (Figure 2A). Moreover, nucleotides G46 and U47 of the V-region are also conserved in all bacterial tRNAAsp (Figure 2A), which mostly, like N.meningitidis tRNAAsp, also display the U12–A23 base pair. These observations indi- cate that the archaeal AdT should not be able to discriminate aspartylated bacterial tRNAAsn and tRNAAsp. However, since all archaeal tRNAAsn possess the G1–C72 base pair shown to prevent amidation by bacterial AdT, one can predict that aspartylated archaeal tRNAAsn should not be a suitable substrate for bacterial AdT. To verify these predictions, we performed cross-species transamidation experiments. tRNAAsn from M.barkeri is aspartylated by T.thermophilus non-discriminating AspRS and the esterifying Asp is amidated by AdT from M.barkeri (Figure 4A). However, as expected, N.meningitidis AdT is unable to promote amidation of Asp bound to the archaeal tRNAAsn (Figure 4A), whereas AdT from M.barkeri amidates Asp bound to both tRNAAsn and tRNAAsp from N.meningitidis (Figure 4B and C). These results conﬁrm that the tRNA elements determining conversion of the esterifying Asp into Asn differ in the bacterial and archaeal systems. They also show that nt 36, C in tRNAAsp and U in tRNAAsn does not constitute a transamidation identity element in the archaeal system.