6086 Nucleic Acids Research, 2006, Vol. 34, No. 21 not allow accurate kinetic measurements. Moreover, another unsolved question is whether AdTs behave similar to Michaelian enzymes or not. Finally, since establishment of kinetic constants for the various aspartylated tRNA variants requires pure aa-tRNA species, the use of in vitro transcribed tRNA species is impossible to circumvent. However, the activity of the GatCAB AdTs studied so far was determined using charged, post-transcriptionally modiﬁed tRNAAsn or tRNAGln species expressed in vivo. Thus the inﬂuence of the tRNA post-transcriptional modiﬁcations on recognition by GatCAB AdT of the tRNA moiety of aa-tRNA remains unknown. The ﬁrst part of this study consisted in the set up of the kinetic measurements of the tRNA-dependent formation of Asn by N.meningitidis AdT. We puriﬁed the enzyme to homogeneity from an E.coli overexpressing strain, and veri- ﬁed that it was not contaminated by measurable traces of E.coli asparagine synthetases A and B catalyzing tRNA- independent Asn formation (Materials and Methods). Since the tRNA mutational investigation rests on the use of a variety of in vitro transcribed tRNAs, we ﬁrst synthesized a N.meningitidis tRNAAsn T7 transcript. The transcript was aspartylated by T.thermophilus non-discriminating AspRS to almost 100% (data not shown). Stability of the ester bond of this Asp-tRNAAsn species was checked by incubation in the transamidation mixture that will subsequently be used. In these conditions the half-life of this aa-tRNA was of 45 min at 37 C (Materials and Methods). We then tes- ted the ability of the [14C] aspartylated tRNAAsn transcript to be amidated in a standard reaction mixture. Incubation of 100 pmol of [14C]Asp-tRNAAsn with 10 pmol of N.meningitidis AdT during 30 min at 37 C resulted in a full conversion into [14C]Asn-tRNAAsn, conﬁrming that aspartylated tRNAAsn transcript deprived of the post- transcriptional modiﬁcations is a suitable substrate for N.meningitidis AdT (data not shown). For kinetic measurements of the transamidation reaction we chose to take time plots ranging from 30 s to 2 min, and conﬁrmed by TCA precipitation of aliquots of the reac- tion mixture that, in agreement with the half-life of the Asp-tRNAAsn, deacylation of the aa-tRNA within this time scale is negligible. Figure 1A shows the TLC analyzing the conversion of [14C]Asp-tRNAAsn into [14C]Asn-tRNAAsn for a range of aa-tRNA concentrations varying from 0.8 to 8 mM. The initial rates derived from this analysis (Materials and Methods) were used to draw the double reciprocal plot (Figure 1B). The linearity of this plot unambiguously proves that N.meningitidis AdT behaves like a Michaelian enzyme. The KM (1.2 mM) and kcat (0.64 s 1) values of N.meningitidis AdT for the cognate aspartylated transcript were determined from two independent measurements. The inﬂuence of the post-transcriptional modiﬁcations of tRNA on the efﬁciency of the AdT-catalyzed reaction was analyzed by comparing the kinetic constants of amidation of Asp acylating either T.thermophilus tRNAAsn, puriﬁed from an E.coli expressing strain, or N.meningitidis T7 tRNAAsn transcript. Since N.meningitidis AdT amidates aspartylated tRNAAsn from T.thermophilus (KM ¼ 0.6 mM, kcat ¼ 0.34 s 1) and N.meningitidis (Table 1) with com- parable efﬁciencies, the post-transcriptional modiﬁcations of tRNAAsn are of minor importance in transamidation, Figure 1. Determination of the kinetic constants of amidation of Asp esteri- fying the tRNAAsn transcript by N.meningitidis AdT. (A) Autoradiogram of the TLC plate analysing the conversion of tRNA-bound [14C]Asp into [14C]Asn catalyzed by 25 nM of pure N.meningitidis AdT as a function of the concentration [14C]Asp-tRNAAsn (mM) and time (s). The concentrations of aa-tRNA are indicated on the top of each series of time plots. The reactions were conducted as described in Materials and Methods. The remaining [14C]Asp and the [14C]Asn formed in 10 ml aliquots after various time intervals are determined by TLC analysis as described in Materials and Methods. (B) Double reciprocal plot: initial rate 1 ¼ f ([Asp-tRNAAsn] 1). The initial rates were determined from amounts of [14C]Asn formed after increasing incubation times at 37 C shown in (A). The intensities of the spots were quantified as described in Materials and Methods. justifying the use of variants of tRNA transcripts for characterizing the tRNA transamidation identity elements. tRNA identity elements for Asp-tRNAAsn transamidation by bacterial AdT The identiﬁcation of the candidate nucleotides of tRNAAsn ensuring efﬁcient transamidation of Asp-tRNAAsn into Asn- tRNAAsn was driven by two assumptions. We ﬁrst assumed that these nucleotides are conserved in the tRNAAsn species of all organisms that use Asp-AdT to form Asn-tRNAAsn, and that the tRNA determinants promoting Asn formation by AdT are not species-speciﬁc. This hypothesis is supported by previous results showing that AdTs from various bacterial origins are able to efﬁciently amidate Asp bound to tRNAAsn from other origins (15,18). The second hypothesis was based on the fact that all AdTs, studied so far, are unable to amidate the other aspartylated tRNA species formed in the cell, namely Asp-tRNAAsp. Therefore, nucleotides con- served in tRNAAsn that can also be found in tRNAAsp should be excluded from set of structural elements potentially responsible for tRNA-dependent Asn formation. We applied these two search criteria on a comparative alignment of 49 bacterial tRNAAsn and tRNAAsp gene sequences. Out of the 8 bp and 9 single nucleotides conserved in all tRNAAsn spe- cies only 1 bp, U1–A72 in tRNAAsn and G1–C72 in tRNAAsp, and 1 nt, U36 in tRNAAsn and C36 in tRNAAsp, matched our criteria (Figure 2A). In addition, tRNAAsp and tRNAAsn also differ by the length of their D-loop. In tRNAAsn, this loop is