a b s t r a c tThe role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl-tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. tRNA-dependent amino acid biosynthesisThe attachment of amino acids to the 3 0 -end of tRNAs is catalyzed by the aminoacyl-tRNA synthetase (aaRS) family of proteins [1]. aaRSs are ubiquitous and essential but only eukaryotes and a handful of bacteria have the full set of 20 enzymes, one for each canonical amino acid in the genetic code. Most bacteria and archaea lack asparaginyl-tRNA synthetase (AsnRS) and/or glutaminyl-tRNA synthetase (GlnRS) and some methanogenic archaea lack cysteinyl-tRNA synthetase (CysRS) [2]. Also, no aaRS for the rare amino acid selenocysteine has been found in any domain of life [3]. These organisms instead use indirect pathways to synthesize a number of amino acids (Asn, Cys, Gln and Sec) directly on their cognate tRNA: non-discriminating aaRSs first form misacylated aminoacyl-tRNA (aa-tRNA), which is not used by the ribosome but instead converted to cognate aa-tRNA by various RNA-dependent modifying enzymes [4].In organisms lacking GlnRS and AsnRS, Glu-tRNA Gln and AsptRNA Asn are synthesized by non-discriminating aaRSs and converted to cognate Gln-tRNA Gln and Asn-tRNA Asn by tRNA-dependent amidotransferases (AdT). Two types of AdT exist, the heterotrimeric GatCAB present in both bacteria and archaea and the homodimeric GatDE present in archaea [5,6]. The tRNA moiety is recognized by the B and E kinase subunits of GatCAB and GatDE, respectively, which phosphorylate the mischarged tRNAs to form activated intermediates [7][8][9]. The glutaminase subunit (GatA/D) liberates ammonia from an amide donor and amidates Glu or Asp on the tRNA to form Gln or Asn, respectively. In both types of AdT, a 40 Å-long hydrophilic channel connects the glutaminase and kinase subunits [9,10]. It has been proposed, but remains to be proven, that ammonia liberated in the glutaminase active site is transported through the channel via a series of protonations and deprotonations to the kinase active site, and that binding of mischarged tRNA is required for opening the channel. Another open question concerns the precise in vivo mechanism by which misacylated aa-tRNA species are stabilized and escape detection, and subsequent delivery to the ribosome, by e...