Non-discriminating aspartyl-tRNA Synthetase Research Articles

Evolution and variation in amide aminoacyl-tRNA synthesis.

The amide proteogenic amino acids, asparagine and glutamine, are two of the twenty amino acids used in translation by all known life. The aminoacyl-tRNA synthetases for asparagine and glutamine, asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase, evolved after the split in the last universal common ancestor of modern organisms. Before that split, life used two-step indirect pathways to synthesize asparagine and glutamine on their cognate tRNAs to form the aminoacyl-tRNA used in translation. These two-step pathways were retained throughout much of the bacterial and archaeal domains of life and eukaryotic organelles. The indirect routes use non-discriminating aminoacyl-tRNA synthetases (non-discriminating aspartyl-tRNA synthetase and non-discriminating glutamyl-tRNA synthetase) to misaminoacylate the tRNA. The misaminoacylated tRNA formed is then transamidated into the amide aminoacyl-tRNA used in protein synthesis by tRNA-dependent amidotransferases (GatCAB and GatDE). The enzymes and tRNAs involved assemble into complexes known as transamidosomes to help maintain translational fidelity. These pathways have evolved to meet the varied cellular needs across a diverse set of organisms, leading to significant variation. In certain bacteria, the indirect pathways may provide a means to adapt to cellular stress by reducing the fidelity of protein synthesis. The retention of these indirect pathways versus acquisition of asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase in lineages likely involves a complex interplay of the competing uses of glutamine and asparagine beyond translation, energetic costs, co-evolution between enzymes and tRNA, and involvement in stress response that await further investigation.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.

Anticodon‐Binding Domain Mutations of the Bacillus subtilis Non‐discriminating Aspartyl‐tRNA Synthetase to Increase tRNA Specificity

The fidelity of the genetic code is etablished by tRNA synthetases attaching an amino acid to the correct tRNA isoacceptor. The majority of prokaryotes lack an asparaginyl‐tRNA synthetase to ligate asparagine to tRNAAsn. Instead, these organisms possess a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS), an AspRS with relaxed substrate specificity, that can ligate aspartate to either tRNAAsn or tRNAAsp. An amidotransferase enzyme, GatCAB, then amidates Asp‐tRNAAsn to form Asn‐tRNAAsn. In many but not all organisms encoding an asparaginyl‐tRNA synthetase to directly ligate asparagine to tRNAAsn, the aspartyl‐tRNA synthetase has evolved to be specific for tRNAAsp, a discriminating aspartyl‐tRNA synthetase (D‐AspRS). How bacterial aspartyl‐tRNA synthetase evolved specificity for tRNAAsp is not clear. Bacillus subtilis encodes a ND‐AspRS while the related Bacillus anthracis codes for a D‐AspRS. The sequence similarity between the two enzymes provides an opportunity to alter the tRNA specificity of a ND‐AspRS. Using the sequence comparison along with available structures of aspartyl‐tRNA synthetases, we have mutated the anticodon binding domain of the B. subtilis ND‐AspRS and will report how the various mutations impacted specificity for tRNAAsp. The work expands our understanding of tRNA synthetase substrate specificity and how fidelity of protein synthesis is achieved.Support or Funding InformationNational Science Foundation (MCB‐1615770)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Å resolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNAAsn

Synthesis of asparaginyl-tRNA (Asn-tRNAAsn) in bacteria can be formed either by directly ligating Asn to tRNAAsn using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNAAsn and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNAGln formation. Most bacteria are predicted to use only one route for Asn-tRNAAsn formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNAAsn formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNAGln synthesis, their AspRS enzymes were thought to be specific for tRNAAsp. However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNAAsp by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code.

Dual Routes for B. subtilis Asparaginyl‐tRNA Formation

Two distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis, are known. The one-step, direct pathway uses an asparaginyl-tRNA synthetase (AsnRS) to aminoacylate Asn to tRNAAsn. In organisms lacking AsnRS, a two-step pathway is used. First a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches aspartate (Asp) to tRNAAsn. The Asp is then amidated to Asn by GatCAB. Despite Bacillus subtilis using AsnRS for Asn-tRNAAsn production and GatCAB for Gln-tRNAGln formation, we predicted the lone B. subtilis AspRS is non-discriminating to enable the organism to also use the two-step pathway for Asn-tRNAAsn formation. Our in vitro and in vivo results are consistent with the AspRS using tRNAAsn as a substrate as the first step in tRNA-dependent Asn biosynthesis. B. subtilis encoding a tRNA-dependent route for Asn production would explain why the organism is able to grow in the absence Asn when all three of its glutamine-dependent Asn synthetases are knocked out. This work was supported by Skidmore College and the National Science Foundation (MCB-1244326)

Two Distinct Routes for Asparaginyl‐tRNA Formation in B. halodurans

In nature, two distinct routes have evolved for attaching the amino acid asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis. In the direct pathway Asn is ligated to tRNAAsn by an asparaginyl-tRNA synthetase (AsnRS). In a second, two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) first misacylates Asp to tRNAAsn. This tRNA-bound Asp is then amidated to Asn by the amidotransferase GatCAB. Bioinformatic analysis shows that the Gram-positive Bacillus halodurans can use the direct route. However, whether it uses the indirect route depends on whether its AspRS is non-discriminating. Using both in vivo and in vitro assays, we demonstrate the B. halodurans AspRS can aspartylate tRNAAsn and can be used with GatCAB to form Asn-tRNAAsn. Aminoacylation of tRNAAsn by AsnRS is five-fold more efficient than with AspRS. Retention of the two-step pathway for Asn-tRNA formation in B. halodurans may be vestigial though is might be advantageous unde...

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

Dual Routes for Bacteriovorus bdellovibrio Asparaginyl‐tRNA Formation

Two distinct paths have evolved in nature to attach the amino acid asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in translation. In the first (the direct pathway), Asn is directly ligated to tRNAAsn by asparaginyl‐tRNA synthetase (AsnRS). In the second (the indirect pathway), a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS) attaches Asp to tRNAAsn, and then GatCAB transamidates the Asp to Asn on the tRNA. Bioinformatic analysis predicts the predatory bacterium Bacteriovorus bdellovibrio codes for both pathways. Our work has focused on whether B. bacteriovorus encodes a ND‐AspRS and GatCAB for tRNA‐dependent Asn biosynthesis. Our studies, using the E. coli Asn auxotrophic strain JF448, demonstrate that together the B. bacteriovorus AspRS and GatCAB can synthesize Asn on tRNA. The non‐discriminating nature of the B. bacteriovorus AspRS is being further verified by using the E. coli TrpA34 system and in vitro studies. We speculate encoding dual routes for Asn‐tRNAAsn formation enables B. bacteriovorus to grow efficiently in the presence and absence of a host.

Dual Routes for Bacillus halodurans Asparaginyl‐tRNA Formation

Two evolutionary distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis, are found in nature. In the first, asparaginyl‐tRNA synthetase directly ligates Asn to tRNAAsn in an ATP‐dependent manner. In the second pathway, a non‐discriminating aspartyl‐tRNA synthetase (ND‐AspRS) first ligates Asp to tRNAAsn and GatCAB then modifies the tRNA‐bound Asp to Asn by transamidating the amino acid. Our bioinformatics analysis of the Gram‐positive Bacillus halodurans suggests it may encode the indirect pathway in addition to the direct one. Encoding the indirect Asn‐tRNAAsn biosynthetic pathway requires an ND‐AspRS. We are testing whether the B. halodurans AspRS is non‐discriminating (i.e., uses tRNAAsn as a substrate along with tRNAAsp). Using the E. coli JF448 system, we have determined the B. halodurans AspRS is non‐discriminating and works in concert with GatCAB to synthesize Asn in a tRNA‐dependent manner. Further verification of the nature of the AspRS using the E. coli TrpA34 system and in vitro studies are underway. We speculate retention of both pathways for Asn‐tRNAAsn synthesis in B. halodurans may be vestigial and that a number of other bacteria may also maintain both routes.

Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements

Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNAAsp and tRNAAsn with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of α-helix and β-sheet secondary structure at 20°C and predominantly β-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and 1H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein.

A tRNA-independent Mechanism for Transamidosome Assembly Promotes Aminoacyl-tRNA Transamidation

Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln). In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNA(Asn) from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNA(Asn). Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a non-discriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNA(Asn) for assembly. Hp0100 also enhances the capacity of AdT to convert Asp-tRNA(Asn) into Asn-tRNA(Asn) by ∼35-fold. Our results demonstrate that bacteria have adopted multiple divergent methods for transamidosome assembly and function.

Mechanism and evolution of multidomain aminoacyl-tRNA synthetases revealed by their inhibition by analogues of a reaction intermediate, and by properties of truncated forms

Many enzymes which catalyze the conversion of large substrates are made of several structural domains belonging to the same polypeptide chain. Transfer RNA (tRNA), one of the substrates of the multidomain aminoacyl-tRNA synthetases (aaRS), is an L-shaped molecule whose size in one dimension is similar to that of its cognate aaRS. Crystallographic structures of aaRS/tRNA complexes show that these enzymes use several of their structural domains to interact with their cognate tRNA. This mini review discusses first some aspects of the evolution and of the flexibility of the pentadomain bacterial glutamyl-tRNA synthetase (GluRS) revealed by kinetic and interaction studies of complementary truncated forms, and then illustrates how stable analogues of aminoacyl-AMP intermediates have been used to probe conformational changes in the active sites of Escherichia coli GluRS and of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) of Pseudomonas aeruginosa.

Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNAAsn amidotransferase activity

Only about half of bacterial species use an asparaginyl-tRNA synthetase (AsnRS) to attach Asn to its cognate tRNA(Asn). Other bacteria, including the human pathogen Moraxella catarrhalis, a causative agent of otitis media, lack a gene encoding AsnRS, and form Asn-tRNA(Asn) by an indirect pathway catalysed by two enzymes: first, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) catalyses the formation of aspartyl-tRNA(Asn) (Asp-tRNA(Asn)); then, a tRNA-dependent amidotransferase (GatCAB) transamidates this 'incorrect' product into Asn-tRNA(Asn). As M. catarrhalis has a Gln-tRNA synthetase, its GatCAB functions as an Asp-tRNA(Asn) amidotransferase. This pathogen rapidly evolved to about 90 % ampicillin resistance worldwide by insertion of a bro-1 β-lactamase gene within the gatCAB operon. Comparison of the GatCAB subunits from bro-1 β-lactamase-positive and bro-negative strains showed that the laterally transferred bro-1 gene, inserted into the gatCAB operon, affected the C-terminal sequence of GatA. The identity between the C-terminal sequences of GatA(wt) (residues 479-491) and of GatA(BRO-1) (residues 479-492) was about 36 %, whereas the rest of the GatA sequence was relatively conserved. The characterization of these two distinct GatCABs as well as the hybrid GatCAB containing GatA(1-478)(wt)(479-492)(BRO-1) and truncated GatCAB enzymes of M. catarrhalis showed that the substitution in GatA(wt) of residues 479-492 of GatA(BRO-1) causes increased specificity for glutamine, and decreased specificity for Asp-tRNA(Asn) in the transamidation reaction. We conclude that the bro gene insertion has altered the kinetic parameters of Asp-tRNA(Asn) amidotransferase, and we propose a model for gatA evolution after the insertion of bro-1 at the carboxyl end of gatA.

The asparagine-transamidosome from Helicobacter pylori : a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code

Helicobacter pylori catalyzes Asn-tRNAAsn formation by use of the indirect pathway that involves charging of Asp onto tRNAAsn 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) tRNAAsn binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNAAsn is bound by ND-AspRS which releases the Asp-tRNAAsn product much slower than the cognate Asp-tRNAAsp; this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNAAsn 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.

Crystal structure of a transfer-ribonucleoprotein particle that promotes asparagine formation

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.

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Archaea make glutaminyl-tRNA (Gln-tRNAGln) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNAGln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNAGln. Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNAAsn) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228–239.]. Here, we show a similar complex for Gln-tRNAGln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNAGln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (KD = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNAGln formation, with GatDE reducing the affinity of ND-GluRS for tRNAGlu by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNAGln) formation, and has no major effect on the kinetics of tRNAGln glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.

Isolation, crystallization and preliminary X-ray analysis of the transamidosome, a ribonucleoprotein involved in asparagine formation

Thermus thermophilus deprived of asparagine synthetase synthesizes Asn on tRNA(Asn) via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNA(Asn) prior to conversion of the Asp to Asn by GatCAB, a tRNA-dependent amidotransferase. This pathway also constitutes the route of Asn-tRNA(Asn) formation by bacteria and archaea deprived of asparaginyl-tRNA synthetase. The partners involved in tRNA-dependent Asn formation in T. thermophilus assemble into a ternary complex called the transamidosome. This particule produces Asn-tRNA(Asn) in the presence of free Asp, ATP and an amido-group donor. Crystals of the transamidosome from T. thermophilus were obtained in the presence of PEG 4000 in MES-NaOH buffer pH 6.5. They belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 115.9, b = 214.0, c = 127.8 A, beta = 93.3 degrees . A complete data set was collected to 3 A resolution. Here, the isolation and crystallization of the transamidosome from T. thermophilus and preliminary crystallographic data are reported.