Published online 29 October 2006 Nucleic Acids Research, 2006, Vol. 34, No. 21 6083–6094 doi:10.1093/nar/gkl622 A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine Marc Bailly, Stamatina Giannouli1, Mickael Blaise, Constantinos Stathopoulos1,*, Daniel Kern* and Hubert Dominique Becker De´ partement ‘Machineries traductionnelles’, UPR 9002 Architecture et Re´ activite´ de l’ARN, Institut de Biologie Mole´ culaire et Cellulaire du CNRS, 15, Rue Rene´ Descartes, F-67084 Strasbourg Ce´ dex, France and 1Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos street, 41221 Larissa, Greece Received July 6, 2006; Revised and Accepted August 8, 2006 ABSTRACT 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 tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. 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 tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1–A72 whereas the G1–C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same com- bination 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 nucleo- tides G46 and U47 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. INTRODUCTION Flawless protein synthesis requires a set of perfectly paired aminoacyl-tRNAs (aa-tRNAs). It was de facto expected that each organism should possess a full complement of aminoacyl-tRNA synthetases (aaRS) capable of matching each amino acid to its cognate tRNA. More recently it became obvious that direct formation of aa-tRNA by charg- ing preformed amino acids on its cognate tRNA with aaRS is not universally conserved (1). In fact, the vast majority of prokaryotes, including almost all prominent human pathogens, use a two-step alternate route to synthesize the aa-tRNAs carrying the amidated amino acid, asparagine (Asn) and glutamine (Gln) (2–4). This pathway that forms the amidated aa-tRNA by transamidation of an aa-tRNA precursor is catalyzed by a tRNA-dependent amidotrans- ferase (AdT). This enzyme supplies the ribosomes of organ- isms lacking asparaginyl-tRNA synthetase (AsnRS) and glutaminyl-tRNA synthetase (GlnRS), with asparaginyl- tRNAAsn (Asn-tRNAAsn) and glutaminyl-tRNAGln (Gln- tRNAGln). In a ﬁrst step, the mischarged aspartyl-tRNAAsn (Asp-tRNAAsn) and glutamyl-tRNAGln (Glu-tRNAGln) are produced by an aspartyl-tRNA synthetase (AspRS) or a glutamyl-tRNA synthetase (GluRS) of relaxed speciﬁcity (3,5). These mischarged aa-tRNA escape protein synthesis since they are not binding to elongation factor Tu (EF-Tu) and therefore are not directed to the ribosomes (3,6). Instead, they are substrates for subsequent transamidation by AdTs, thus forming the correctly paired Asn-tRNAAsn and Gln-tRNAGln that will serve for decoding Asn and Gln codons on the ribosome. In vivo, bacterial AdTs ensure formation of either Asn-tRNAAsn (Asp-AdT) or Gln-tRNAGln (Glu-AdT) or both aa-tRNAs (Asp/Glu-AdT) depending on the genomic context of the organism. However, in vitro, all bacterial AdTs studied so far exhibit dual speciﬁcity and do not discriminate between the two mischarged aa-tRNAs spe- cies. They form both, Asn-tRNAAsn and Gln-tRNAGln, using the mischarged Asp-tRNAAsn and Glu-tRNAGln as substrates. Furthermore, the AdT enzymes exhibit different oligomeric structures in bacteria and archaea. Bacteria solely use a heterotrimeric enzyme (GatCAB) encoded by the gatC, gatA and gatB genes. This enzyme which can amidate both Asp attached to tRNAAsn and Glu to tRNAGln is also encoded *To whom correspondence should be addressed. Tel: +33 3 88 41 70 92; Fax: +33 3 88 60 22 18; Email: d.kern@ibmc.u-strasbg.fr *Correspondence may also be addressed to Constantinos Stathopoulos. Tel: +30 2410 56 52 78; Fax: +30 2410 56 52 90; Email: cstath@bio.uth.gr 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.