Hubert Dominique Becker

Yeast mitochondrial Gln-tRNA Gln is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS

It is impossible to predict which pathway, direct glutaminylation of tRNA(Gln) or tRNA-dependent transamidation of glutamyl-tRNA(Gln), generates mitochondrial glutaminyl-tRNA(Gln) for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA(Gln) has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA(Gln) 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 ((c)ERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA(Gln) substrate for the AdT. We show that dual localization of (c)ERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for (c)ERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of (c)ERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA(Gln) 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.

When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

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-tRNAAsn 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.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants

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-tRNAGln 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-tRNAGln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNAGln, which is the only tRNAGln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNAGln 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-tRNAGln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.

The heterotrimeric Thermus thermophilus Asp-tRNAAsn amidotransferase can also generate Gln-tRNAGln

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl‐tRNAAsn amidotransferase (Asp‐AdT) capable of forming Asn‐tRNAAsn [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln‐tRNAGln as well as Asn‐tRNAAsn. Thus, the T. thermophilus tRNA‐dependent AdT is a dual‐specific Asp/Glu‐AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy‐terminal amino acids of the GatA subunit only inhibits the Asp‐AdT activity, leaving the Glu‐AdT activity of the mutant AdT unaltered; this shows that Asp‐AdT and Glu‐AdT activities can be mechanistically separated.

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

In most prokaryotes Asn-tRNAAsn and Gln-tRNAGln are formed by amidation of aspartate and glutamate mischarged onto tRNAAsn and tRNAGln, respectively. Coexistence in the organism of mischarged Asp-tRNAAsn and Glu-tRNAGln and the homologous Asn-tRNAAsn and Gln-tRNAGln 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-tRNAAsn 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-tRNAAsn from Asn-tRNAAsn and Asp-tRNAAsp 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.

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, tRNAAsn 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 tRNAsAsn 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‐tRNAAsn 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.

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

The gatC, gatA and gatB genes encoding the three subunits of glutamyl‐tRNAGln 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‐tRNAGln to Gln‐tRNAGln and Asp‐tRNAAsn to Asn‐tRNAAsn. 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.

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

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-tRNAGln 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 Å and a docking model of the synthetase complexed to tRNAGln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNAGln 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 tRNAGln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNAGln 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.

Two-codon T-box riboswitch binding two tRNAs

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 tRNAAsn and tRNAGlu. 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.

Translating organellar glutamine codons A case by case scenario

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-tRNAGln (Q-tRNAQ) 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-tRNAQ 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-tRNAQ synthesis in yeast and mammals mitochondria is still questionable.