Aminoacyl-tRNA Synthesis by Pre-Translational Amino Acid Modification

[RNA Biology 1:1, 16-20; May/June 2004]; ©2004 Landes Bioscience

Aminoacyl-tRNA Synthesis by Pre-Translational Amino Acid Modification Review

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ABSTRACT

Aminoacyl-tRNAs (aa-tRNAs) are essential substrates for ribosomal translation, and are generally synthesized by aminoacyl-tRNA synthetases (aaRSs). It was expected earlier that every organism would contain a complete set of twenty aaRSs, one for each canonical amino acid. However, analysis of the many known genome sequences and biochemical studies revealed that most organisms lack asparaginyl- and glutaminyl-tRNA synthetases, and thus are unable to attach asparagine and glutamine directly onto their corresponding tRNA. Instead, a pretranslational amino acid modification is required to convert Asp-tRNAAsn and Glu-tRNAGln to the correctly charged Asn-tRNAAsn and Gln-tRNAGln, respectively. This transamidation pathway of amide aa-tRNA synthesis is common in most bacteria and archaea. Unexpected results from biochemical, genetic and genomic studies showed that a large variety of different bacteria rely on tRNA-dependent transamidation for the formation of the amino acid asparagine. Pretranslational modifications are not restricted to asparagine and glutamine but are also found in the biosynthesis of some other aa-tRNAs, such as the initiator tRNA fmet-tRNAMeti and Sec-tRNASec specifying selenocysteine, the 21st cotranslationally inserted amino acid. tRNA-dependent amino acid modification is also involved in the generation of aminolevulinic acid, the first precursor for porphyrin biosynthesis in many organisms.

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*Correspondence to: Dieter Söll; Department of Molecular Biophysics and Biochemistry; Yale University; P.O. Box 208114; 266 Whitney Avenue; New Haven, Connecticut 06520-8114 USA; Tel.: 203.432.6200; Fax: 203.432.6202; Email: [email protected]

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address: Medical University of South Carolina; Department of Biochemistry and Molecular Biology; Charleston, South Carolina 29412 USA

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1Departments of Molecular Biophysics and Biochemistry; 2Department of Chemistry; Yale University; New Haven, Connecticut USA

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Liang Feng1 Kelly Sheppard1 Suk Namgoong1 Alexandre Ambrogelly1 Carla Polycarpo1 Lennart Randau1 Debra Tumbula-Hansen1,† Dieter Söll1,2,*

Received 04/05/04; Accepted 04/28/04

INTRODUCTION

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Previously published online as a RNA Biology E-publication: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=953

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KEY WORDS

Faithful translation of the genetic information relies on the precise matching of the mRNA codons with their corresponding amino acids according to the rules of the genetic code. As first proposed by Crick,1 an adaptor molecule is required as an interface between the messenger RNA and the polypeptide chain being synthesized. This adaptor molecule, known as tRNA for transfer RNA, delivers the amino acid attached at its 3’ end to the ribosome for protein synthesis by paring specifically with the codon on the mRNA. The overall fidelity of gene expression therefore depends on the correct attachment of an amino acid onto its tRNA catalyzed by the corresponding aaRS. Aminoacyl-tRNA formation is a two-step process. The amino acid (aa) is first activated in the presence of ATP to form the aminoacyl-adenylate (aa-AMP, step 1). The activated amino acid is then transferred onto the 2’- or 3’-hydroxyl group of the 3’ terminal adenosine of the cognate tRNA molecule (tRNAaa) with the release of AMP (step 2). aa + ATP + aaRS aaRS • aa-AMP + PPi (1) aaRS • aa-AMP + tRNAaa aa-tRNAaa + AMP + aaRS (2) To date, all twenty aaRS enzymes, one for each canonical amino acid, have been identified. Interestingly, these enzymes fall into two evolutionarily unrelated classes based on distinct active site topologies which impose different mechanistic features on ATP recognition, tRNA binding and regio-specificity of the transfer reaction.2,3 Structural studies have shown that the catalytic core of all class I aaRSs (enzymes specific for leucine, isoleucine, methionine, valine, tyrosine, tryptophan, glutamic acid, glutamine, cysteine, argininine, and sometimes lysine) resembles the Rossmann dinucleotide-binding fold, while the active site of class II enzymes (those specific for serine, proline, threonine, alanine, histidine, aspartic acid, asparagine, lysine, phenylalanine, and glycine) contains an antiparallel β-fold with three degenerate sequence motifs designated as motifs I, II and III.4-7 All the aaRS orthologs that are specific for a given amino acid, with the exception of lysine,8 belong to the same class of aaRS across the three domains of life. Until a few years ago it was generally thought that a complete set of twenty aaRSs are required for cell survival. However, with the availability of many genomic sequences of

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aminoacyl-tRNA, aminoacyl-tRNA synthetase, amidotransferase, selenocysteine, pyrrolysine

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ACKNOWLEDGEMENTS

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Work in the authors’ laboratory was supported by grants from the National Institute of General Medical Sciences and the Department of Energy.

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very diverse organisms from all three domains of life, it became clear that only the eukaryotic cytoplasm and some bacteria contain the whole family of aaRS proteins. Most prokaryotes and organelles in fact survive without a full set of aaRSs.9 Among those often missing are glutaminyltRNA synthetase (GlnRS) and asparaginyl-tRNA synthetase (AsnRS). Instead, an alternative pathway via transamidation of misaminoacylated Asp-tRNAAsn and Glu-tRNAGln is utilized to synthesize the corresponding Asn-tRNAAsn and Gln-tRNAGln. This biosynthetic route of amide aa-tRNA formation by pretranslational modification bears resemblances to the biosyntheses of fMet-tRNAMeti and Sec-tRNASec, and possibly pyrrolysyl-tRNAPyl (Fig. 1).10-15 This article summarizes our current understanding of aa-tRNA synthesis by tRNA-dependent amino acid modification.

WIDESPREAD PRESENCE OF tRNA-DEPENDENT TRANSAMIDATION

Transamidation of Glu-tRNAGln to Gln-tRNAGln was first discovered thirty-five years ago in Bacillus subtilis and related species.16 These Gram-positive bacteria cannot charge glutamine directly onto tRNAGln and therefore Figure 1. Indirect routes to asparaginyl-, glutaminyl-, formylmethionyl-, and selenocysteyl-tRNA using an aaRS with relaxed tRNA specificity and a tRNA-dependent amino acid modification rely on a tRNA-dependent amidotransferase (AdT) enzyme. AspRS, aspartyl-tRNA synthetase; GluRS, glutamyl-tRNA synthetase; MetRS, Gln to convert a misacylated tRNA, Glu-tRNA to methionyl-tRNA synthetase; SerRS, seryl-tRNA synthetase; AspAdT, aspartyl-tRNAAsn amidotransGln-tRNAGln for translation16 (Fig. 1). Later, an ferase; GluAdT, glutamyl-tRNAGln amidotransferase; MTF, methionyl-tRNA formyltransferase; SelA, analogous pathway for indirect Asn-tRNAAsn selenocysteine synthase. formation via transamidation of Asp-tRNAAsn was also demonstrated17 (Fig. 1). The misaminoacylated tRNAAsn late sixties that GluRS exits in a nondiscriminating (ND) form in and tRNAGln are synthesized by the corresponding aspartyl-tRNA B. subtilis.16 Unlike the discriminating GluRS found in E. coli and synthetase (AspRS) and glutamyl-tRNA synthetase (GluRS) with the eukaryal cytoplasm that recognizes tRNAGlu only, this nondisrelaxed tRNA specificity (e.g., refs. 18 and 19, also see below). criminating GluRS (ND-GluRS) possesses a relaxed tRNA specificity The widespread presence of tRNA-dependent transamidation and can charge glutamate onto either tRNAGlu or tRNAGln. A only became apparent recently with the availability of a large number nondiscriminating AspRS (ND-AspRS) that synthesizes both of genomic sequences.9 Analysis of fully sequenced microbial Asp-tRNAAsp and Asp-tRNAAsn was later identified in both archaeal genomes revealed that the majority lack recognizable GlnRS and and bacterial domains.18,19,22 Similarly, the formation of AsnRS homologs, but have to rely on the indirect pathway for the fMet-tRNAMeti and Sec-tRNASec also require specificity-relaxed genetic decoding of asparagine and glutamine. To date, no GlnRS SerRS and MetRS, respectively.11,12 has been identified in any archaeal organism.9,20 A canonical GlnRS The molecular basis for the relaxed tRNA specificity of these is only present in a minority of bacteria such as Thermus thermophilus unusual enzymes is still in its infancy, but an understanding of the and Deinococcus radiodurans, the green sulfur bacteria, clostridium, dual tRNA selection of archaeal AspRS enzymes is emerging.23,24 and some proteobacteria2,9,18 (Fig. 2). We have recently demonstrated that a single amino acid change in All archaea sequenced to date except those of the Pyrococcus and the tRNA anticodon binding loop of AspRS converted the discrimThermoplasma/Ferroplasma groups lack a canonical AsnRS inating Thermococcus kodakaraensis AspRS to a nondiscriminating (http://www.tigr.org/tdb/).20-22 The absence of AsnRS is also common enzyme that also recognizes tRNAAsn.23 Whether the reverse mutain bacteria, as this enzyme is missing from many Gram-positive tion could limit the tRNA recognition of a ND-AspRS to tRNAAsp bacteria, proteobacteria of the α, β and ε subdivisions, and the should shed light on the evolutionary divergence of ancestral AspRS Chlamydiales as well as the two deeply rooted division Aquificales enzymes and the emergence of the direct asparaginylation pathway. and Thermotogales9 (Fig. 2).

HOW ASN-tRNAASN AND GLN-tRNAGLN ARE FORMED

tRNA-DEPENDENT AMINO ACID TRANSFORMATION REQUIRES NONDISCRIMINATING SYNTHETASES WITH RELAXED tRNA SPECIFICITY

Biochemical examination of aaRS enzymes showed already in the www.landesbioscience.com

The significance of the initial discovery of tRNA-dependent transamidation was not fully appreciated, and further characterization of the reaction was also hindered by the lack of understanding of the enzymes involved. The situation changed when the B. subtilis

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do not share homology with any other protein. The other subunits of the two AdTs are unrelated. GatA belongs to the large family of amidases characterized by the amidase signature sequence, a stretch of ~100aa rich in glycine and serine residues.30 GatD is similar to Type I asparaginase.20 Since both amidases and asparaginases catalyze the reaction of amide bond hydrolysis, GatA and GatD are likely responsible for releasing the active ammonia from amide donor like glutamine for transamidation. Despite the similar chemistry, the two enzyme families have completely different tertiary structures and active site topologies. Members of the amidase family contain a novel Ser-cis Ser-Lys catalytic triad that is not found in the asparaginases.20,30 The function of the small ~10 kD GatC subunit is unclear, but it may serve as a chaperon for GatA based on some earlier results.25 The existence of two alternate and redundant routes to Asn-tRNAAsn and Gln-tRNAGln allowed an organism the freedom of choice. Biochemical data and genome analysis of bacteria reveal four different scenarios, all of which are based on the presence of only one operational pathway for the formation of either Gln-tRNAGln or Asn-tRNAAsn (Fig. 2). (1) Organisms lacking AsnRS and GlnRS using Asp/Glu-AdT: A large set of bacteria (e.g., Chlamydia trachomatis) lacks these two synthetases. Instead, the bacterial Asp/Glu-AdT GatCAB carries out both functions.19,27 Obviously, this requires that both GluRS and AspRS be nondiscriminating. (2) Bacteria with GlnRS but lacking AsnRS: Some organisms (e.g., Pseudomonas aeroginosa; ref. 31) lack a ND-GluRS and employ GlnRS for Gln-tRNAGln formation while Asp/Glu-AdT GatCAB supplies Asn-tRNAAsn. (3) Bacteria with AsnRS but lacking GlnRS: Here is the opposite situation in which AsnRS forms Asn-tRNAAsn and GatCAB provides Gln-tRNAGln Figure 2. Bacterial distribution of direct and indirect pathways for Asn-tRNAAsn and (e.g., Bacillus subtilis). (4) Organisms with AsnRS and Gln-tRNAGln synthesis. The occurrence of genes encoding AsnRS, GlnRS and Asp/Glu-AdT in genome sequences from representative bacterial groups is indicated. GlnRS: Some γ-proteobacteria (e.g., E. coli) acquired 32 The bacterial phylogeny is based upon 16S rRNA, adapted from GlnRS from eukaryotes by horizontal gene transfer and Asn Gln therefore produce Asn-tRNA and Gln-tRNA by http://www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi. direct acylation. The reasons for this unexpected diversity AdT was first cloned and characterized.25 The heterotrimeric of amide aa-tRNA formation will be a challenge to research. Glu-tRNAGln-dependent AdT (GluAdT) is encoded by the gatC, gatA, and gatB genes. Their arrangement is operonic (gatCAB) in OTHER tRNA-DEPENDENT AMINO ACID MODIFICATIONS some organisms (e.g., B. subtilis), while in other organisms (e.g., tRNA-dependent pretranslational modifications are not unique Helicobacter pylori) the genes are dispersed in the genome. It was a surprise to learn that the conversion of Asp-tRNAAsn to for asparagine and glutamine, as cotranslational insertions of the Asn-tRNAAsn is catalyzed in vivo also by the same GatCAB initiator formylmethionine and rare amino acids such as selenoenzyme.19,26-27 The complex structure of the GatCAB enzyme cysteine and possibly pyrrolysine all require tRNA-dependent explained early difficulties in cloning and purification of the protein. modifications.10-15 In bacteria and eukaryal organelles, protein Now the gat genes have been identified in many bacteria, archaea, synthesis requires a specific formylmethionyl-tRNAfMet (fMetand organelles, and recombinant GatCAB enzyme has been purified tRNAfMet) as the initiator aminoacyl-tRNA. The formation of this from numerous sources.19,20,25,29 A different heterodimeric AdT particular fMet-tRNAfMet also follows an indirect route. Methionine encoded by the gatD and gatE genes was later identified in archaea.20 is first charged onto tRNAfMet by methionyl-tRNA synthetase (MetRS) The two AdTs not only have distinct oligomeric structures, but and is then formylated by a highly specific methionyl-tRNA formylalso differ in tRNA recognition and natural distribution. The transferase (MTF) in the presence of the formyl donor N-formylteheterotrimeric GatCAB is dual specific for Asp-tRNAAsn and trahydrofolate2,10-11 (Fig. 1). Glu-tRNAGln, and this Asp/Glu-AdT is ubiquitously distributed Synthesis of selenocysteyl-tRNA follows a similar mechanism as among the three domains of life.9 In contrast, GatDE is restricted to the indirect amide aminoacyl-tRNA formation, but with a different the archaeal domain and recognizes Glu-tRNAGln only.20 However, chemistry.2,12-13 Serine is first attached to a selenocysteine-specific the two AdTs are evolutionarily related as GatE shares homology to tRNA, tRNASec, by a seryl-tRNA synthetase (SerRS) (Fig. 1). This GatB. The function of these subunits remains undetermined as they misacylated Ser-tRNASec is further converted to Sec-tRNASec by 18

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selenocysteine synthase (SelA). The SelA reaction also requires selenophosphate, which is synthesized by selenophosphate synthetase (SelD). The Sec-tRNASec is transported to the ribosome by the special elongation factor SelB for insertion of the rare amino acid at an internal UGA codon. The so-called 22nd amino acid, pyrrolysine, was recently found to be inserted into the UAG codons in monomethylamine methyltransferases of Methanosarcina barkeri.14 The biosynthetic pathway of pyrrolysyl-tRNA is not yet well understood and maybe brought about by the uncharacterized PylS protein14 or by a combination of both LysRS I and LysRS II.15 However, a two step tRNA-dependent amino acid transformation could exist for pyrrolysine, analogous to those just described. This Lys-tRNAPyl may be the substrate for a further modification enzyme (e.g., PylS) to produce Pyl-tRNAPyl.15 Alternatively, if pyrrolysine were a regular metabolite, PylS may directly charge it onto tRNAPyl (see ref. 33).

tRNA-DEPENDENT ASPARAGINE BIOSYNTHESIS

In addition to their central role in protein synthesis, aminoacyltRNAs also function in a variety of other metabolic functions. In particular, aminoacyl-tRNAs appear to be involved in tRNA-dependent biosynthesis of asparagine and glutamine in many bacteria and some archaea18,29,34 (Fig. 1). In E. coli, asparagine is synthesized by transamidation of free aspartic acid by either an ammonium-dependent asparagine synthetase (AsnA) or a glutamine-dependent asparagine synthetase (AsnB). However, fully sequenced bacterial genomes of thirteen different genera representing most of the main bacterial groups have no homologs of AsnA and AsnB but contain an AspAdT,34 suggesting a role of this enzyme in asparagine biosynthesis in these organisms in addition to its role in Asn-tRNAAsn formation.18,29,31 This proposal could be tested genetically if one could obtain a viable strain through inactivation of the AspAdT pathway, and observe its growth phenotype in medium lacking asparagine. Such experiments were possible in Deinococcus radiodurans, an asparagine prototrophic bacteria that lacks both asparagine synthetases but possesses redundant pathways for Asn-tRNAAsn formation.18,29 Asn-tRNAAsn in Deinococcus radiodurans could be synthesized either directly via AsnRS or indirectly by transamidaiton via AspAdT. Disruption of the AspAdT pathway in D. radiodurans therefore did not lead to a lethal phenotype since Asn-tRNAAsn could still be made by the AsnRS in the mutant strain.34 Rather, D. radiodurans was converted to an asparagine auxotroph upon inactivation of the AspAdT pathway through deletion of the AspRS with the relaxed tRNA specificity.34 Furthermore, introduction of the AspAdT pathway of D. radiodurans into an asparagine auxotrophic E. coli strain enabled the recombinant strain to grow on an asparagine-free medium,34 suggesting that the transamidation pathway is sufficient for asparagine biosynthesis. Whether free intracellular asparagine, if required, is the result of natural deacylation of the Asn-tRNAAsn or is the product of an enzymatic reaction catalyzed by a specific aminoacyl-tRNA hydrolase remains to be investigated. On the other hand, no free asparagine may be needed in a cell if asparagine is only needed for protein synthesis. In contrast to bacteria, out of sixteen archaea with completed genome sequences, only 2, Sulfolobus solfataricus and Methanosarcina acetivorans, seem to depend on AspAdT for asparagine biosynthesis (ref. 35 and www.ncbi.nlm.nih.gov/genomes/MICROBES/ Complete.html). The rest of them contain an AsnB homolog and could obtain asparagine directly from free aspartate. www.landesbioscience.com

Glutamine synthetase (encoded by glnA), which synthesizes glutamine from glutamate and ammonium, is almost universally present and plays a critical role in controlling nitrogen metabolism. However, glutamine synthetase appears to be missing in a few bacterial groups such as the chlamydias, spirochetes and mycoplasmas. The presence of a GluAdT in these glnA lacking organisms could suggest a possible recruitment, as for asparagine, of tRNA-dependent transamidation for glutamine biosynthesis.19 However, the inactivation of glutamine synthetase in Mycobacterium tuberculosis36 and Corynebacterium glutamicum37 provoked a glutamine auxotrophic phenotype. These results indicate that when glnA and GluAdT coexist, tRNA-dependent glutamine formation probably plays a minor role in glutamine biosynthesis, contrasting with organisms lacking glnA completely.

GLUTAMYL-tRNA IS A PRECURSOR IN THE BIOSYNTHESES OF CHLOROPHYLL AND HEME

There is one tRNA-dependent amino acid modification whose reaction product is not destined for protein, but forms the first precursor in porphyrin biosynthesis.38 In this case Glu-tRNAGlu in chloroplasts and many bacteria is converted by glutamyl-tRNA reductase (GluTR)—encoded by the hemA gene—in a NADHdependent reaction to glutamate 1-semialdehyde.39 This compound is then transformed into the ‘recognized’ first precursor of the porphyrin ring system, 5-aminolevulinic acid in a pyridoxal phosphate-dependent transamination.38 The tRNA serves two purposes in the GluTR reaction, not only activating the α-carbonyl group of glutamate for catalysis but also providing a good leaving group. As both GluTR and GluRS recognize the same tRNA, it was interesting to see that there is no overlap in the tRNAGlu identity elements for both enzymes (Randau L, personal communication).

FINAL REMARK

Given the diverse mechanisms and occurrences of tRNA-dependent amino acid transformations involved in aa-tRNA synthesis, many evolutionary questions come to mind. Why is the formation of Gln-tRNAGln not firmly integrated throughout the living world? Are asparagine and glutamine the last amino acids added to the repertoire of canonical amino acids found in proteins? Are GlnRS and AsnRS the last aaRS proteins to evolve? Was there a link between amino acid and protein synthesis? Only further research will satisfy our curiosity. References 1. Crick FH. On protein synthesis. Symp Soc Exp Biol 1953; 12:138-63. 2. Ibba M, Söll D. Aminoacyl-tRNA synthesis. Annu Rev Biochem 2000; 69:617-50. 3. Eriani G, Delarue M, Poch O, Gangloff J, Moras D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 1990; 347:203-6. 4. Cusack S, Berthet-Colominas C, Härtlein M, Nassar N, Leberman R. A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 Å. Nature 1990; 347:249-55. 5. Moras D. Structural and functional relationships between aminoacyl-tRNA synthetases. Trends Biochem Sci 1992; 17:159-64. 6. Cusack S. Sequence, structure and evolutionary relationships between class 2 aminoacyl-tRNA synthetases: An update. Biochimie 1993; 75:1077-81. 7. Arnez JG, Moras D. Structural and functional considerations of the aminoacylation reaction. Trends Biochem Sci 1997; 22:211-6. 8. Ibba M, Morgan S, Curnow AW, Pridmore DR, Vothknecht UC, Gardner W, et al. A euryarchaeal lysyl-tRNA synthetase: Resemblance to class I synthetases. Science 1997; 278:1119-22. 9. Feng L, Tumbula-Hansen D, Min B, Namgoong S, Salazar J, Orellana O, et al. Transfer RNA-dependent amidotransferases: Key enzymes for Asn-tRNA and Gln-tRNA synthesis in nature. In: Ibba M., Francklyn C, Cusack S, eds. The Aminoacyl-tRNA Synthetases. Georgetown, TX: Landes Biosciences, 2004.

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10. Blanquet S, Mechulam Y, Schmitt E. The many routes of bacterial transfer RNAs after aminoacylation. Curr Opin Struct Biol 2000; 10:95-101. 11. Mayer C, Stortchevoi A, Köhrer C, Varshney U, RajBhandary UL. Initiator tRNA and its role in initiation of protein synthesis. Cold Spring Harb Symp Quan Biol 2001; 66:195-206. 12. Rother M, Resch A, Wilting R, Böck A. Selenoprotein synthesis in archaea. Biofactors 2001; 14:75-83. 13. Hatfield DL, Gladyshev VN. How selenium has altered our understanding of the genetic code. Mol Cell Biol 2002; 22:3565-76. 14. Srinivasan G, James CM, Krzycki JA. Pyrrolysine encoded by UAG in Archaea: Charging of a UAG-decoding specialized tRNA. Science 2002; 296:1459-62. 15. Polycarpo C, Ambrogelly A, Ruan B, Tumbula-Hansen D, Ataide SF, Ishitani R, et al. Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases. Mol Cell 2003; 12:287-94. 16. Wilcox M, Nirenberg M. Transfer RNA as a cofactor coupling amino acid synthesis with that of protein. Proc Natl Acad Sci USA 1968; 61:229-36. 17. Curnow AW, Ibba M, Söll D. tRNA-dependent asparagine formation. Nature 1996; 382:589-90. 18. Becker HD, Kern D. Thermus thermophilus: A link in evolution of the tRNA-dependent amino acid amidation pathways. Proc Natl Acad Sci USA 1998; 95:12832-7. 19. Raczniak G, Becker HD, Min B, Söll D. A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis. J Biol Chem 2001; 276:45862-7. 20. Tumbula DL, Becker HD, Chang WZ, Söll D. Domain-specific recruitment of amide amino acids for protein synthesis. Nature 2000; 407:106-10. 21. Woese CR, Olsen GJ, Ibba M, Söll D. Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process. Microbiol Mol Biol Rev 2000; 64:202-36. 22. Tumbula-Hansen D, Feng L, Toogood H, Stetter KO, Söll D. Evolutionary divergence of the archaeal aspartyl-tRNA synthetases into discriminating and nondiscriminating forms. J Biol Chem 2002; 277:37184-90. 23. Feng L, Tumbula-Hansen D, Toogood H, Söll D. Expanding tRNA recognition of a tRNA synthetase by a single amino acid change. Proc Natl Acad Sci USA 2003; 100:5676–5681. 24. Charron C, Roy H, Blaise M, Giegé R, Kern D. Nondiscriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain. EMBO J 2003; 22:1632-43. 25. Curnow AW, Hong K, Yuan R, Kim S, Martins O, Winkler W, et al. Glu-tRNAGln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc Natl Acad Sci USA 1997; 94:11819-26. 26. Becker HD, Min B, Jacobi C, Raczniak G, Pelaschier J, Roy H, et al. The heterotrimeric Thermus thermophilus Asp-tRNAAsn amidotransferase can also generate Gln-tRNAGln. FEBS Lett 2000; 476:140-4. 27. Salazar JC, Zuniga R, Raczniak G, Becker HD, Söll D, Orellana OA. Dual-specific Glu-tRNAGln and Asp-tRNAAsn amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans. FEBS Lett 2001; 500:129-31. 28. Horiuchi KY, Harpel MR, Shen L, Luo Y, Rogers KC, Copeland RA. Mechanistic studies of reaction coupling in Glu-tRNAGln amidotransferase. Biochemistry 2001; 40:6450-7. 29. Curnow AW, Tumbula DL, Pelaschier JT, Min B, Söll D. Glutamyl-tRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis. Proc Natl Acad Sci USA 1998; 95:12838-43. 30. Shin S, Lee TH, Ha NC, Koo HM, Kim SY, Lee HS, et al. Structure of malonamidase E2 reveals a novel Ser-cisSer-Lys catalytic triad in a new serine hydrolase fold that is prevalent in nature. EMBO J 2002; 21:2509-16. 31. Akochy PM, Bernard D, Roy PH, Lapointe J. Direct glutaminyl-tRNA biosynthesis and indirect asparagingyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1. J Bacteriol 2004; 186:767-76. 32. Lamour V, Quevillon S, Diriong S, N’Guyen VC, Lipinski M, Mirande M. Evolution of the Glx-tRNA synthetase family: The glutaminyl enzyme as a case of horizontal gene transfer. Proc Natl Acad Sci USA 1994; 91:8670-4. 33. Ibba M, Söll D. Aminoacyl-tRNAs: Setting the limits of the genetic code. Genes Dev 2004; 18:731-8. 34. Min B, Pelaschier JT, Graham DE, Tumbula-Hansen D, Söll D. Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation. Proc Natl Acad Sci USA 2002; 99:2678-83. 35. Roy H, Becker HD, Reinbolt J, Kern D. When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism. Proc Natl Acad Sci USA 2003; 100:9837-42. 36. Tullius MV, Harth G, Horwitz MA. Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs. Infect Immun 2003; 71:3927-36. 37. Jakoby M, Tesch M, Sahm H, Kramer R, Burkovski A. Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I. FEMS Microbiol Lett 1997; 154:81-8. 38. Kumar AM, Schaub U, Söll D, Ujwal ML. Glutamyl-transfer RNA: At the crossroad between chlorophyll and protein biosynthesis. Trends Plant Sci 1996; 1:371-6. 39. Schauer S, Chaturvedi S, Randau L, Moser J, Kitabatake M, Lorenz S, et al. Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate. J Biol Chem 2002; 277:48657-63.

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