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 tRNA^Asn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNA^Asn formation that GatCAB can then amidate to asparaginyl-tRNA^Asn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNA^Asn 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-tRNA^Asn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.     

Aminoacylation of Plasmodium falciparum tRNAAsn and Insights in the Synthesis of Asparagine Repeats

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA^Asn. However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA^Asn accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA^Asn in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as “tRNA sponges” and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.     

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 tRNA^Asp and tRNA^Asn generating a correctly charged Asp-tRNA^Asp and an erroneous Asp-tRNA^Asn. 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 tRNA^Asp over tRNA^Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNA^Asp and was unable to aspartylate tRNA^Asn. 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.     

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Archaea make glutaminyl-tRNA (Gln-tRNA^Gln) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA^Gln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA^Gln. Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA^Asn) 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-tRNA^Gln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA^Gln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K_D = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA^Gln formation, with GatDE reducing the affinity of ND-GluRS for tRNA^Glu 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-tRNA^Gln) formation, and has no major effect on the kinetics of tRNA^Gln 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.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

On the evolution of the tRNA-dependent amidotransferases, GatCAB and GatDE

Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNA^Gln and asparaginyl-tRNA^Asn were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNA^Gln and aspartyl-tRNA^Asn, by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNA^Gln that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNA^Asn, suggesting that tRNA^Asn recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.     

A dual-specific Glu-tRNA and Asp-tRNA amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans

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.     

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 Å 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.     

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNA^Phe, tRNA^Tyr, tRNA ₁ ^Met , tRNA^Asn, tRNA^Ala, tRNA^Leu, and tRNA^Lys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.We respectfully dedicate this paper to Prof. H. Bauer on the occasion of his 80th birthday.     

A missense mutation in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase suppresses a mitochondrial tRNA(Asp) mutation

The nuclear suppressor allele NSM3 in strain FF1210-6C/170-E22 (E22), which suppresses a mutation of the yeast mitochondrial tRNA^Asp gene in Saccharomyces cerevisiae, was cloned and identified. To isolate the NSM3 allele, a genomic DNA library using the vector YEp13 was constructed from strain E22. Nine YEp13 recombinant plasmids were isolated and shown to suppress the mutation in the mitochondrial tRNA^Asp gene. These nine plasmids carry a common 4.5-kb chromosomal DNA fragment which contains an open reading frame coding for yeast mitochondrial aspartyl-tRNA synthetase (AspRS) on the basis of its sequence identity to the MSD1 gene. The comparison of NSM3 DNA sequences between the suppressor and the wild-type version, cloned from the parental strain FF1210-6C/170, revealed a G to A transition that causes the replacement of amino acid serine (AGU) by an asparagine (AAU) at position 388. In experiments switching restriction fragments between the wild type and suppressor versions of the NSM3 gene, the rescue of respiratory deficiency was demonstrated only when the substitution was present in the construct. We conclude that the base substitution causes the respiratory rescue and discuss the possible mechanism as one which enhances interaction between the mutated tRNA^Asp and the suppressor version of AspRS.     

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

On the role of an unusual tRNAGly isoacceptor in Staphylococcus aureus

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.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA^Gln, that AspRS is nondiscriminating, and that its Asp-tRNA^Asn product is transamidated by AdT. On the other hand, tRNA^Gln is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

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 tRNA^Asn and tRNA^Gln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNA^Asn and Gln-tRNA^Gln. 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 tRNA^Asn and tRNA^Asp variants, that amidation of Asp acylating tRNA^Asn is promoted by the base pair U₁–A₇₂ whereas the G₁–C₇₂ pair and presence of the supernumerary nucleotide U_20A in the D-loop of tRNA^Asp prevent amidation. We predict, based on comparison of tRNA^Gln and tRNA^Glu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNA^Gln, 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.     

Riboswitch (T-box)-mediated control of tRNA-dependent amidation in Clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-trnaasn synthesis

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.     

Characterization of a New CAMP Factor Carried by an Integrative and Conjugative Element in Streptococcus agalactiae and Spreading in Streptococci

Genetic exchanges between Streptococci occur frequently and contribute to their genome diversification. Most of sequenced streptococcal genomes carry multiple mobile genetic elements including Integrative and Conjugative Elements (ICEs) that play a major role in these horizontal gene transfers. In addition to genes involved in their mobility and regulation, ICEs also carry genes that can confer selective advantages to bacteria. Numerous elements have been described in S. agalactiae especially those integrated at the 3′ end of a tRNA^Lys encoding gene. In strain 515 of S. agalactiae, an invasive neonate human pathogen, the ICE (called 515_tRNA^Lys) is functional and carries different putative virulence genes including one encoding a putative new CAMP factor in addition to the one previously described. This work demonstrated the functionality of this CAMP factor (CAMP factor II) in Lactococcus lactis but also in pathogenic strains of veterinary origin. The search for co-hemolytic factors in a collection of field strains revealed their presence in S. uberis, S. dysgalactiae, but also for the first time in S. equisimilis and S. bovis. Sequencing of these genes revealed the prevalence of a species-specific factor in S. uberis strains (Uberis factor) and the presence of a CAMP factor II encoding gene in S. bovis and S. equisimilis. Furthermore, most of the CAMP factor II positive strains also carried an element integrated in the tRNA^Lys gene. This work thus describes a CAMP factor that is carried by a mobile genetic element and has spread to different streptococcal species.