2-mercaptoethanol, 0.1 mM benzamidine and Na2EDTA and 50% glycerol. Wild-type, transcribed and mutated tRNAAsp and tRNAAsn from N.meningitidis, T.thermophilus and total tRNA from M.barkeri For in vitro transcription, wild-type and mutated N.meningitidis and T.thermophilus tRNAAsp and tRNAAsn genes were cloned under control of the T7 RNA polymerase promoter using the cassette-cloning procedure (16). The species beginning with 50U were cloned downstream the sequence of a self-cleaving ribozyme (17). Transcription, self-cleavage and puriﬁcation of the transcripts were per- formed as described previously (16). The transcripts exhib- ited 60–100% of accepting capacity. For in vivo expression, the T.thermophilus tRNAAsn gene was extended with the 50 GAT triplet and the 30 AATTCAAA octanucleotide to mimic the genomic context of E.coli tRNAAsn gene and cloned into the pKK223 vector (16). The tRNAAsn overex- pressed in E.coli JM 103 strain was puriﬁed, after phenol extraction of the nucleic acids, by chromatographies on DEAE–cellulose, Sepharose 4B, Octyl Sepharose 4B and Hydroxyapatite. Pure tRNAAsn (18 mg) (accepting capacity, 36 nmol/mg) was obtained from 200 g of cells. Unfraction- ated tRNAs from M.barkeri were isolated from nucleic acids extracted by shaking the cell suspension (6 g cells in 6 ml of 50 mM sodium acetate buffer, pH 5.1, containing 1 mM MgCl2 and 10 mM Na2EDTA) brieﬂy sonicated with 1 vol of acid-buffered phenol overnight at room temperature. DNA and long RNAs were precipitated by addition of 20% isopropanol to the aqueous layer recovered by centrifugation. The tRNAs recovered by DEAE–cellulose chromatography were precipitated with 60% isopropanol and dissolved in water. Preparation of [14C]Asp-tRNAs The standard aminoacylation mixture (50–200 ml) containing 100 mM Na–HEPES, pH 7.2, 30 mM KCl, 2 mM ATP, 12 mM MgCl2, 10–100 mM L-[14C]Asp (300 cpm/pmol), 2– 10 mM tRNA or transcripts and 0.2–1 mM of T.thermophilus non-discriminating AspRS was incubated 10 min at 50 C. The reaction was stopped by acid-buffered phenol and chloroform extractions; the [14C]Asp-tRNA was precipitated with ethanol in the aqueous layer recovered by centrifugation, sedimented, re-dissolved in water and the concentration was determined by scintillation counting of aliquots after TCA precipitation. tRNA-dependent amidation assays The standard reaction mixture of 50 ml containing 100 mM Na–HEPES buffer, pH 7.2, 12 mM MgCl2, 2 mM L-Asn, 1 mM ATP, 40–400 pmol [14C]Asp-tRNA and appropriate amounts of N.meningitidis or T.thermophilus AdT or 5 ml of M.barkeri GatCAB enriched fraction was incubated 30 min at 37 C. The kinetic constants were measured in the standard reaction mixture containing 0.8–8 mM [14C]Asp- tRNA and 25 or 250 nM of AdT when KM or kcat/KM was measured, respectively. After incubation times ranging from 0 to 240 s, 10 ml aliquots were withdrawn, supplemented with 40 ml of water and mixed with phenol–chloroform Nucleic Acids Research, 2006, Vol. 34, No. 21 6085 to stop the reaction. When pure AdT was used, the [14C]aa-tRNA present in the aqueous layer recovered by centrifugation, was deacylated by 30 min incubation at 80 C in the presence of 25 mM KOH, followed by neutraliza- tion with HCl. When an AdT-enriched M.barkeri protein fraction was used the [14C]aa-tRNA was precipitated after phenol–chloroform extraction with ethanol to remove the traces of free Asn that could have been formed by contami- nating asparagine synthetases, dissolved in 50 ml of water and deacylated as described above. The hydrolysate was dried in a Speed-Vac, dissolved in 3 ml of water and fraction- ated by TLC on cellulose plates (20 · 20 cm2) extended by a 3MM Whatman paper sheet (20 · 5 cm2), with a solvent containing 2-propanol/formic acid/water (80:4:20, by vol). The [14C] amino acids were revealed by scanning the 2 h exposed image plate with a Fuji Bioimager. The half-lifes of wild-type and mutated [14C]Asp-tRNAAsn (45 min) were determined from deacylation kinetics con- ducted by TCA precipitation after various incubation times of 10 ml aliquots of the amidation mixtures deprived of AdT followed by scintillation counting. Since the kinetics of [14C]Asp-tRNAAsn amidation were conducted within 4 min, the decrease of the concentration of [14C]Asp-tRNA promoted by deacylation was negligible. However, since [14C]Asn-tRNAAsn is less stable (12 min) the kinetics of formation of [14C]Asn-tRNAAsn were established without precipitation of the aa-tRNA by determining the total, free and tRNA-bound, [14C]Asn. Quantification of the [14C]Asn The intensities of the [14C]Asn spots were quantiﬁed using the volume rectangle tool of the Quantity One software (BIO-RAD). The intensity units of each spot were corrected by subtraction of the background intensity and converted into pmoles of [14C] amino acids by determining, for each TLC plate, the speciﬁc activity (Intensity Units/pmol) of the [14C]aa. This value is an average of at least ﬁve deter- minations and was determined from ratio of the intensity units of the spot over the amount (pmol) of [14C]aa-tRNA present. RESULTS AND DISCUSSION Establishment of the kinetic behavior of N.meningitidis AdT The aim of this work was to identify the structural elements of tRNA responsible for speciﬁc amidation of Asp acylating tRNAAsn into Asn. This investigation requires synthesis of a series of tRNAAsn mutants and the use of an assay that allows monitoring of the effect of the mutations on the tRNA-dependent Asn synthesis. For this purpose we chose to measure the effect of tRNA mutations on the efﬁciency on the AdT-catalyzed Asp amidation by establishing the kinetic constants of the aspartylated tRNA variants. However, the amidation assay (Materials and Methods) used to analyze AdT activity has, so far, never been applied to kinetic mea- surements. The absence of kinetic studies on GatCAB AdTs is mainly due to the instability of the ester bond between amino acid and tRNA of the aa-tRNA substrate which might