A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNA(Asn) and tRNA(Gln). These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA(Asn) and Gln-tRNA(Gln). So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNA(Asn) and tRNA(Asp) variants, that amidation of Asp acylating tRNA(Asn) is promoted by the base pair U(1)–A(72) whereas the G(1)–C(72) pair and presence of the supernumerary nucleotide U(20A) in the D-loop of tRNA(Asp) prevent amidation. We predict, based on comparison of tRNA(Gln) and tRNA(Glu) sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA(Gln), that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G(46) and U(47) of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.