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The objective of the present review is to provide an insight into modifications of microbial cell walls and membrane constituents by using the aminoacyl-tRNA as amino acid donor. In bacteria, phospholipids are modified by Multiple peptide resistance Factor enzymes and peptidoglycan precursors by so called fem ligases. Although these modifications were thought to be restricted to procaryotes, we discovered enzymes that modify ergosterol (the main component of fungal membrane) with glycine and aspartate. The focus of this review is to present the molecular mechanisms underlying all these processes together with the structure of the enzymes and their substrates. This article also reviews how substrates are recognized and modified and how the products are subsequently exported in various organisms. Finally, the physiological outcome and the discoveries of each family of enzymes is also discussed.

Genetic research; Nervous system diseases; Pediatrics Investigación genética; Enfermedades del sistema nervioso; Pediatría Recerca genètica; Malalties del sistema nerviós; Pediatria Background Aminoacyl-tRNA synthetases (ARS) are key enzymes catalysing the first reactions in protein synthesis, with increasingly recognised pleiotropic roles in tumourgenesis, angiogenesis, immune response and lifespan. Germline mutations in several ARS genes have been associated with both recessive and dominant neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase encoded by seryl-tRNA synthetase 1 (SARS1) were reported. Methods We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, developmental delay and seizures, but without microcephaly. Complementation and serylation assays using patient’s fibroblasts and an Saccharomyces cerevisiae model were performed to examine this variant’s pathogenicity. Results A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised interleukin-6, p21, p16 and p53 levels). Conclusion We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence. This study was supported by grants from the Undiagnosed Rare Disease programme URD-Cat (SLT002/16/00174) from the Generalitat de Catalunya, the Center for Biomedical Research on Rare Diseases (CIBERER) (ACCI19-759), the ASL-HSP France and the Hesperia Foundation to AP. This study was also funded by the Instituto de Salud Carlos III and ‘Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, una manera de hacer Europa’ (FIS PI20/00758) to CC and ‘La Marató de TV3’ Foundation (202006-30) to CC and AP. This study was also funded by Instituto de Salud Carlos III (Sara Borrell programme, CD19/00221) to EV, the Spanish Ministerio de Economia, Industria y Competividad (Juan de la Cierva programme FJCI-2016-28811) to EV, and the Center for Biomedical Research on Rare Diseases (CIBERER) to MR and NL. This study was supported by the French National Programme Investissement d’Avenir administered by the ‘Agence National de la Recherche’ (ANR), ‘MitoCross’ Laboratory of Excellence (Labex), funded as ANR-10-IDEX-0002-02, the University of Strasbourg and CNRS (to HB, BS). The CERCA Programme/Generalitat de Catalunya provided institutional support. AP is member of the Undiagnosed Disease Network International (UDNI).

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.

The aminoacylation reaction is one of most extensively studied cellular processes. The so-called "canonical" reaction is carried out by direct charging of an amino acid (aa) onto its corresponding transfer RNA (tRNA) by the cognate aminoacyl-tRNA synthetase (aaRS), and the canonical usage of the aminoacylated tRNA (aa-tRNA) is to translate a messenger RNA codon in a translating ribosome. However, four out of the 22 genetically-encoded aa are made "noncanonically" through a two-step or indirect route that usually compensate for a missing aaRS. Additionally, from the 22 proteinogenic aa, 13 are noncanonically used, by serving as substrates for the tRNA- or aa-tRNA-dependent synthesis of other cellular components. These nontranslational processes range from lipid aminoacylation, and heme, aa, antibiotic and peptidoglycan synthesis to protein degradation. This chapter focuses on these noncanonical usages of aa-tRNAs and the ways of generating them, and also highlights the strategies that cells have evolved to balance the use of aa-tRNAs between protein synthesis and synthesis of other cellular components.

The aminoacylation reaction is one of most extensively studied cellular processes. The so-called “canonical” reaction is carried out by direct charging of an amino acid (aa) onto its corresponding transfer RNA (tRNA) by the cognate aminoacyl-tRNA synthetase (aaRS), and the canonical usage of the aminoacylated tRNA (aa-tRNA) is to translate a messenger RNA codon in a translating ribosome. However, four out of the 22 genetically-encoded aa are made “noncanonically” through a two-step or indirect route that usually compensate for a missing aaRS. Additionally, from the 22 proteinogenic aa, 13 are noncanonically used, by serving as substrates for the tRNA- or aa-tRNA-dependent synthesis of other cellular components. These nontranslational processes range from lipid aminoacylation, and heme, aa, antibiotic and peptidoglycan synthesis to protein degradation. This chapter focuses on these noncanonical usages of aa-tRNAs and the ways of generating them, and also highlights the strategies that cells have evolved to balance the use of aa-tRNAs between protein synthesis and synthesis of other cellular components.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

Prokaryotic and eukaryotic cytosolic aminoacyl-tRNA synthetases (aaRSs) are essentially known for their conventional function of generating the full set of aminoacyl-tRNA species that are needed to incorporate each organism's repertoire of genetically-encoded amino acids during ribosomal translation of messenger RNAs. However, bacterial and eukaryotic cytosolic aaRSs have been shown to exhibit other essential nonconventional functions. Here we review all the subcellular compartments that prokaryotic and eukaryotic cytosolic aaRSs can reach to exert either a conventional or nontranslational role. We describe the physiological and stress conditions, the mechanisms and the signaling pathways that trigger their relocation and the new functions associated with these relocating cytosolic aaRS. Finally, given that these relocating pools of cytosolic aaRSs participate to a wide range of cellular pathways beyond translation, but equally important for cellular homeostasis, we mention some of the pathologies and diseases associated with the dis-regulation or malfunctioning of these nontranslational functions.

Mutations in genes encoding aminoacyl‐tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl‐tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl‐tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole‐exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild‐type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear‐encoded lysine‐rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.

Aminoacylated ergosterol such as 1-ergosteryl aspartate (Erg-Asp) is a new lipid component recently discovered in fungi. In order to study physiological functions of this novel sterol derivative and to develop potential antifungal agents, we established the method to synthesize aminoacylated ergosterol derivatives. Herein, we report the synthesis of Erg-Asp as well as some other aminoacylated ergosterols (Erg-Gly, Erg-Ala, Erg-Leu, Erg-Ile, and Erg-Val) using Boc protected amino acids.

During translational elongation, aminoacylated tRNA are supplied to the ribosome by RNA-interacting proteins. Cex1, a HEAT-containing protein, has been shown to participate to this tRNA channeling by interacting with aminoacylated tRNA during their export from the nucleus. Here, we show that Cex1 is a component of COPI (coatomer complex I) coated vesicles involved in the Golgi-to-vacuole trafficking pathway in Saccharomyces cerevisiae. Cex1 interacts with key components of the COPI coat Sec27, Sec28 and Sec33 proteins. Moreover, fluorescent microscopy indicates that Cex1 is not localized at the nuclear periphery as expected for an effector of nuclear tRNA channeling, but is observed on endosomal trans-Golgi network (TGN) positive structures. This localization relies on the vacuolar protein sorting receptor Vps10. Our data not only resolve the functional ambiguity regarding Cex1 homologues across species, but also point to the possibility to develop yeast-based models to study neurodegenerative disorders linked to the Human Cex1 homologue SCYL1.

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code.

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

In Staphylococcus aureus, a T-box riboswitch exists upstream of the glyS gene to regulate transcription of the sole glycyl-tRNA synthetase, which aminoacylates five tRNAGly isoacceptors bearing GCC or UCC anticodons. Subsequently, the glycylated tRNAs serve as substrates for decoding glycine codons during translation, and also as glycine donors for exoribosomal synthesis of pentaglycine peptides during cell wall formation. Probing of the predicted T-box structure revealed a long stem I, lacking features previously described for similar T-boxes. Moreover, the antiterminator stem includes a 42-nt long intervening sequence, which is staphylococci-specific. Finally, the terminator conformation adopts a rigid two-stem structure, where the intervening sequence forms the first stem followed by the second stem, which includes the more conserved residues. Interestingly, all five tRNAGly isoacceptors interact with S. aureus glyS T-box with different binding affinities and they all induce transcription readthrough at different levels. The ability of both GCC and UCC anticodons to interact with the specifier loop indicates ambiguity during the specifier triplet reading, similar to the unconventional reading of glycine codons during protein synthesis. The S. aureus glyS T-box structure is consistent with the recent crystallographic and NMR studies, despite apparent differences, and highlights the phylogenetic variability of T-boxes when studied in a genome-dependent context. Our data suggest that the S. aureus glyS T-box exhibits differential tRNA selectivity, which possibly contributes toward the regulation and synchronization of ribosomal and exoribosomal peptide synthesis, two essential but metabolically unrelated pathways.

The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA Glu as substrate in vitro nor in vivo , but aminoacylates tRNA Asp with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA Asp is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organellar GluRSs (α-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis .

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNA Asn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.

In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control.

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and ancient enzymes, mostly known for their essential role in generating aminoacylated tRNAs. During the last two decades, many aaRSs have been found to perform additional and equally crucial tasks outside translation. In metazoans, aaRSs have been shown to assemble, together with non-enzymatic assembly proteins called aaRSs-interacting multifunctional proteins (AIMPs), into so-called multi-synthetase complexes (MSCs). Metazoan MSCs are dynamic particles able to specifically release some of their constituents in response to a given stimulus. Upon their release from MSCs, aaRSs can reach other subcellular compartments, where they often participate to cellular processes that do not exploit their primary function of synthesizing aminoacyl-tRNAs. The dynamics of MSCs and the expansion of the aaRSs functional repertoire are features that are so far thought to be restricted to higher and multicellular eukaryotes. However, much can be learnt about how MSCs are assembled and function from apparently ‘simple’ organisms. Here we provide an overview on the diversity of these MSCs, their composition, mode of assembly and the functions that their constituents, namely aaRSs and AIMPs, exert in unicellular organisms.

Yeast mitochondrial Gln-mtRNAGln is synthesized by the transamidation of mischarged Glu-mtRNAGln by a non-canonical heterotrimeric tRNA-dependent amidotransferase (AdT). The GatA and GatB subunits of the yeast AdT (GatFAB) are well conserved among bacteria and eukaryota, but the GatF subunit is a fungi-specific ortholog of the GatC subunit found in all other known heterotrimeric AdTs (GatCAB). Here we report the crystal structure of yeast mitochondrial GatFAB at 2.0 A resolution. The C-terminal region of GatF encircles the GatA-GatB interface in the same manner as GatC, but the N-terminal extension domain (NTD) of GatF forms several additional hydrophobic and hydrophilic interactions with GatA. NTD-deletion mutants displayed growth defects, but retained the ability to respire. Truncation of the NTD in purified mutants reduced glutaminase and transamidase activities when glutamine was used as the ammonia donor, but increased transamidase activity relative to the full-length enzyme when the donor was ammonium chloride. Our structure-based functional analyses suggest the NTD is a trans-acting scaffolding peptide for the GatA glutaminase active site. The positive surface charge and novel fold of the GatF-GatA interface, shown in this first crystal structure of an organellar AdT, stand in contrast with the more conventional, negatively charged bacterial AdTs described previously.

Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.

The formation of aminoacyl-transfer RNA is a crucial step in ensuring the accuracy of protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how archaea and some bacteria synthesize Asn-tRNA and Gln-tRNA. These amide aminoacyl-tRNAs can be formed by the direct acylation of tRNA, catalysed by asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, respectively. A separate, indirect pathway involves the formation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino acids while they are bound to tRNA, which is catalysed by amidotransferases. Here we show that all archaea possess an archaea-specific heterodimeric amidotransferase (encoded by gatD and gatE) for Gln-tRNA formation. However, Asn-tRNA synthesis in archaea is divergent: some archaea use asparaginyl-tRNA synthetase, whereas others use a heterotrimeric amidotransferase (encoded by the gatA, gatB and gatC genes). Because bacteria primarily use transamidation, and the eukaryal cytoplasm uses glutaminyl-tRNA synthetase, it appears that the three domains use different mechanisms for Gln-tRNA synthesis; as such, this is the only known step in protein synthesis where all three domains have diverged. Closer inspection of the two amidotransferases reveals that each of them recruited a metabolic enzyme to aid its function; this provides direct evidence for a relationship between amino-acid metabolism and protein biosynthesis.

Mitochondria originate from the α-proteobacterial domain of life. Since this unique event occurred, mitochondrial genomes of protozoans, fungi, plants and metazoans have highly derived and diverged away from the common ancestral DNA. These resulting genomes highly differ from one another, but all present-day mitochondrial DNAs have a very reduced coding capacity. Strikingly however, ATP production coupled to electron transport and translation of mitochondrial proteins are the two common functions retained in all mitochondrial DNAs. Paradoxically, most components essential for these two functions are now expressed from nuclear genes. Understanding how mitochondrial translation evolved in various eukaryotic models is essential to acquire new knowledge of mitochondrial genome expression. In this review, we provide a thorough analysis of the idiosyncrasies of mitochondrial translation as they occur between organisms. We address this by looking at mitochondrial codon usage and tRNA content. Then, we look at the aminoacyl-tRNA-forming enzymes in terms of peculiarities, dual origin, and alternate function(s). Finally we give examples of the atypical structural properties of mitochondrial tRNAs found in some organisms and the resulting adaptive tRNA-protein partnership.

T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon–anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum , the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA Asn and tRNA Glu . To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that “flexible” T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.

The glutaminyl-tRNA synthetase (GlnRS) from the radiation-resistant bacterium Deinococcus radiodurans differs from known GlnRSs and other tRNA synthetases by the presence of an additional C-terminal domain resembling the C-terminal region of the GatB subunit of tRNA-dependent amidotransferase (AdT). This atypical synthetase was overexpressed in Escherichia coli, purified and crystallized in the presence of PEG 3350. Orthorhombic crystals were obtained that belong to space group P2(1)2(1)2(1) and diffract to 2.3 A resolution. The crystal structure was solved by molecular replacement using the structure of E. coli GlnRS as a search model.

Analysis of the Gram-positive Clostridium acetobutylicum genome reveals an inexplicable level of redundancy for the genes putatively involved in asparagine (Asn) and Asn-tRNA(Asn) synthesis. Besides a duplicated set of gatCAB tRNA-dependent amidotransferase genes, there is a triplication of aspartyl-tRNA synthetase genes and a duplication of asparagine synthetase B genes. This genomic landscape leads to the suspicion of the incoherent simultaneous use of the direct and indirect pathways of Asn and Asn-tRNA(Asn) formation. Through a combination of biochemical and genetic approaches, we show that C. acetobutylicum forms Asn and Asn-tRNA(Asn) by tRNA-dependent amidation. We demonstrate that an entire transamidation pathway composed of aspartyl-tRNA synthetase and one set of GatCAB genes is organized as an operon under the control of a tRNA(Asn)-dependent T-box riboswitch. Finally, our results suggest that this exceptional gene redundancy might be interconnected to control tRNA-dependent Asn synthesis, which in turn might be involved in controlling the metabolic switch from acidogenesis to solventogenesis in C. acetobutylicum.

International audience; Four out of the 22 aminoacyl-tRNAs (aa-tRNAs) are systematically or alternatively synthesized by an indirect, two-step route requiring an initial mischarging of the tRNA followed by tRNA-dependent conversion of the non-cognate amino acid. During tRNA-dependent asparagine formation, tRNA(Asn) promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-tRNA from non-discriminating aspartyl-tRNA synthetase active site to the GatCAB amidotransferase site. The crystal structure of the Thermus thermophilus transamidosome determined at 3 A resolution reveals a particle formed by two GatCABs, two dimeric ND-AspRSs and four tRNAs(Asn) molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl-tRNA(Asn) without dissociation of the complex. We propose that the crystal structure represents a transient state of the transamidation reaction. The transamidosome constitutes a transfer-ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine.

International audience; Accurate synthesis of aminoacyl-tRNAs (aa-tRNA) by aminoacyl-tRNA synthetases (aaRS) is an absolute requirement for errorless decoding of the genetic code and is studied since more than four decades. In all three kingdoms of life aaRSs are capable of assembling into multi-enzymatic complexes that are held together by auxiliary non-enzymatic factors, but the role of such macromolecular assemblies is still poorly understood. In the yeast Saccharomyces cerevisiae, Arc1p holds cytosolic methionyl-tRNA synthetase ((c)MRS) and glutamyl-tRNA synthetase ((c)ERS) together and plays an important role in fine tuning several cellular processes like aminoacylation, translation and carbon source adaptation.

It is impossible to predict which pathway, direct glutaminylation of tRNAGln or tRNA-dependent transamidation of glutamyl-tRNAGln, generates mitochondrial glutaminyl-tRNAGln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNAGln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNAGln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNAGln substrate for the AdT. We show that dual localization of cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of cERS to satisfy a higher demand of mitochondrial glutaminyl-tRNAGln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.

International audience; Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but glutaminyl-tRNA (Q-tRNA) is an exception to this rule. Glutaminyl-tRNA(Gln) (Q-tRNA(Q)) is formed by this direct pathway in the eukaryotic cytosol and in a small subset of bacteria, but is formed by an indirect transamidation pathway in most bacteria and archaea. To date it is almost impossible to predict what pathway generates organellar Q-tRNA(Q) in a given eukaryote. All eukaryotic genomes sequenced so far, display a single glutaminyl-tRNA synthetase (QRS) gene which is at least responsible for the cytosolic QRS activity, as well as a gene coding for a mitochondrial ortholog of the essential GatB subunit of the tRNA-dependent amidotransferase (AdT). Indeed, QRS activity was found in protozoan mitochondria while AdT activity was characterized in plant organelles. The pathway for Q-tRNA(Q) synthesis in yeast and mammals mitochondria is still questionable.

International audience; In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA(Gly) isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA(Gly) genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA(Gly) isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA(Gly) molecules (including the "pseudogene-encoded" tRNA(Gly)) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the "pseudo"-tRNA(Gly(UCC)) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA(Gly) molecules to form ternary complexes with activated S. aureus EF-Tu.GTP. These tRNA(Gly) molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA(Gly) isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.

International audience; Glutamyl-queuosine tRNA(Asp) synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA(Asp). Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA(Glu), Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3' accepting end of the cognate tRNA(Glu), Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA(Asp). In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS.Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA(Asp) among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA(Asp) that determine recognition by Glu-Q-RS. The analyses made on tRNA(Asp) and tRNA(Asn) show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.

In some living organisms the 20 aa-tRNA species participating in protein synthesis are not charged by a complete set of 20 aminoacyl-tRNA synthetases. In prokaryotes, the deficiency of asparaginyl- and/or glutaminyl-tRNA synthetases is compensated by another aminoacyl-tRNA synthetase of relaxed specificity that mischarges the orphan tRNA and by an enzyme that converts the amino acid into that homologous to the tRNA. In Thermus thermophilus Asn-tRNA(Asn) is formed indirectly via a two-step pathway whereby tRNA(Asn) is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase. The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNA(Asn) promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome. This article deals with the methods and techniques employed to clone the genes encoding the enzymes and the tRNA involved in this pathway, to express them in Escherichia coli, to isolate them on a large scale, and to transcribe and produce mg quantities of pure tRNA(Asn)in vitro. The approaches designed especially for this system include (i) clustering of the ORFs encoding the subunits of the heterotrimeric GatCAB that are sprinkled in the genome into an artificial operon, and (ii) the self-cleavage of the tRNA(Asn) transcript starting with U in 5' position through fusion with a hammerhead ribozyme. Further, the crystallization of the free enzymes is described and the characterization of their assembly with tRNA(Asn) into a ribonucleoprotein particle, as well as the investigation of the catalytic mechanism of Asn-tRNA(Asn) formation by the complex are reported.

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA Gln is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA Gln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA Gln , which is the only tRNA Gln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA Gln by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA Gln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.

Unlike other glutaminyl-tRNA synthetases, the one from radioresistant bacterium Deinococcus radiodurans (Dr- GlnRS) possesses an additional C-terminal extension of 220 residues that shares some homology with the subunit of another enzyme of the translation machinery. Dr-GlnRS has been crystallized in an orthorhombic space group. The crystals diffract X-rays to a resolution of ∼2 A. The determination of the structure of this atypical GlnRS showed that its N- and C-terminal appendices, which encompass in total one third of the protein's 852 amino acids, are actually disordered in the crystal lattice. This example demonstrates that macromolecule crystallization can tolerate large flexible regions in the solvent channels as long as they do not interfere with the packing contacts. This intriguing case is analyzed and discussed in light of current crystallogenesis strategies. cally changed our way of performing structural investigations. 1 Looking at sequence databases often leads to the identification of intriguing protein variants that are appealing targets for structural biology investigation. We recently got interested in an atypical form of glutaminyl-tRNA synthetase from the radio- resistant bacterium Deinococcus radiodurans (Dr-GlnRS). Un- like other members of the GlnRS family, it possesses an additional C-terminal extension of 220 residues that shares some homology with another enzyme of the translation machinery. To get an insight into the function of this extra appendix, we crystallized Dr-GlnRS and solved its structure. Although the crystals did diffract to a resolution of ∼2 A, the C-terminus as well as the N-terminal tail and two internal loops could not be observed in the electron density map. We discuss here an extreme case of disorder (35% of the protein) in well-diffracting crystals.

International audience; Asparagine, one of the 22 genetically encoded amino acids, can be synthesized by a tRNA-dependent mechanism. So far, this type of pathway was believed to proceed via two independent steps. A nondiscriminating aspartyl-tRNA synthetase (ND-DRS) first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase (AdT), which then converts the tRNA-bound aspartate into asparagine. We show herein that the ND-DRS, tRNAAsn, and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process. Our results indicate that the tRNAAsn-mediated linkage between the ND-DRS and AdT enables channeling of the mischarged aspartyl-tRNAAsn intermediate between DRS and AdT active sites to prevent challenging of the genetic code integrity. We propose that formation of a ribonucleoprotein is a general feature for tRNA-dependent amino acid biosynthetic pathways that are remnants of earlier stages when amino acid synthesis and tRNA aminoacylation were coupled.

p 1-11; International audience; Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA(Gln) formation. To analyze the peculiarities of the structure-function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3 A and a docking model of the synthetase complexed to tRNA(Gln) constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA(Gln) are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA(Gln) recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA(Gln) acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.

International audience; In most prokaryotes Asn-tRNA(Asn) and Gln-tRNA(Gln) are formed by amidation of aspartate and glutamate mischarged onto tRNA(Asn) and tRNA(Gln), respectively. Coexistence in the organism of mischarged Asp-tRNA(Asn) and Glu-tRNA(Gln) and the homologous Asn-tRNA(Asn) and Gln-tRNA(Gln) does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA(Asn) intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA(Asn) from Asn-tRNA(Asn) and Asp-tRNA(Asp) by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.

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 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 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.

International audience; Analysis of the completed genome sequences revealed presence in various bacteria of an open reading frame (ORF) encoding a polypeptide chain presenting important similarities with the catalytic domain of glutamyl-tRNA synthetases but deprived of the C-terminal anticodon-binding domain. This paralog of glutamyl-tRNA synthetases, the YadB protein, activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNA(Glu) but instead on tRNA(Asp). It has been shown that tRNA(Asp) is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged by YadB. The functional properties of YadB contrast with those of the canonical glutamyl-tRNA synthetases, which activate Glu only in presence of the cognate tRNA before aminoacylation of the 3'-end of tRNA. Biochemical approaches and mass spectrometry investigations revealed that YadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nucleoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamyl-queuosine. Unstability of the ester bond between the glutamate residue and the cyclopenthene-diol (half-life 7.5 min) explains why until now this modification escaped detection. Among Escherichia coli tRNAs containing queuosine in the wobble position, only tRNA(Asp) is substrate of YadB. Sequence comparison reveals a structural mimicry between the anticodon-stem and loop of tRNA(Asp) and the amino acid acceptor-stem of tRNA(Glu). YadB, renamed glutamyl-Q-tRNA(Asp) synthetase, constitutes the first enzyme structurally related to aminoacyl-tRNA synthetases which catalyzes a hypermodification in tRNA, and whose function seems to be conserved among prokaryotes. The discovery of glutamyl-Q-tRNA(Asp) synthetase breaks down the current paradigm according to which the catalytic domain of aminoacyl-tRNA synthetases recognizes the amino acid acceptor-stem of tRNA and aminoacylates the 3'-terminal ribose. The evolutionary significance of the existence of an aminoacyl-tRNA synthetase paralog dedicated to the hypermodification of a tRNA anticodon will be discussed.

International audience; Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes.

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5Å using molecular replacement. The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS). It is much shorter than GluRS, which contains 468 residues, and lacks the complete domain interacting with the tRNA anticodon loop. As E.coli GluRS, YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain. The YadB cluster uses cysteine residues as the first three zinc ligands, but has a weaker tyrosine ligand at the fourth position. It shares with canonical amino acid RNA synthetases a major functional feature, namely activation of the amino acid (here glutamate). It differs, however, from GluRSs by the fact that the activation step is tRNA-independent and that it does not catalyze attachment of the activated glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA. These results suggest thus a novel function, distinct from that of GluRSs, for the yadB gene family.

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.

Lysyl-tRNA synthesis is catalyzed by two unrelated families of aminoacyl-tRNA synthetases. In most bacteria and all eukarya, the known lysyl-tRNA synthetases (LysRSs) are subclass IIb-type aminoacyl-tRNA synthetases, whereas many archaea and a scattering of bacteria contain an unrelated class I-type LysRS. Examination of the recognition of partially modified tRNA Lys anticodon variants by a bacterial (from Borrelia burgdorferi ) and an archaeal (from Methanococcus maripaludis ) class I lysyl-tRNA synthetase revealed differences in the pattern of anticodon recognition between the two enzymes. U35 and U36 were both important for recognition by the B. burgdorferi enzyme, whereas only U36 played a role in recognition by M. maripaludis LysRS. Examination of the phylogenetic distribution of class I LysRSs suggested a correlation between recognition of U35 and U36 and the presence of asparaginyl-tRNA synthetase (AsnRS), which also recognizes U35 and U36 in the anticodon of tRNA Asn . However, the class II LysRS of Helicobacter pylori , an organism that lacks AsnRS, also recognizes both U35 and U36, indicating that the presence of AsnRS has solely influenced the phylogenetic distribution of class I LysRSs. These data suggest that competition between unrelated aminoacyl-tRNA synthetases for overlapping anticodon sequences is a determinant of the phylogenetic distribution of extant synthetase families. Such patterns of competition also provide a basis for the two separate horizontal gene transfer events hypothesized in the evolution of the class I lysyl-tRNA synthetases.

As originally postulated in Crick's Adaptor hypothesis, the faithful synthesis of proteins from messenger RNA is dependent on the presence of perfectly acylated tRNAs. The hypothesis also suggested that each aminoacyl-tRNA would be made by a unique enzyme. Recent data have now forced a revision of this latter point, with an increasingly diverse array of enzymes and pathways being implicated in aminoacyl-tRNA synthesis. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origins.

Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria.