Two oligomeric types of glycyl-tRNA synthetase (GlyRS) are found in nature: a α2 type and a α2β2 type. The former has been identified in all three kingdoms of life and often pairs with tRNAGly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNAGly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNAGly isoacceptors in both compartments possess A73. We showed herein that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both α2-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1 - strain, while Escherichia coli GlyRS (a α2β2-type enzyme) and A. thaliana organellar GlyRS (a (αβ)2-type enzyme) failed to rescue either defect of the yeast mull allele. However, a head-to-tail αβ fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a α2-type eukaryotic GlyRS may be functionally substituted with a α2β2-type bacterial cognate enzyme despite their remote evolutionary relationships.

WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases and play roles in tRNA or protein binding. We present evidence herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. The cytoplasmic form possessed an Nterminal WHEP domain, whereas its mitochondrial isoform possessed an extra N-terminal sequence consisting of an mitochondrial targeting signal and an appended domain. Cross-species complementation assays showed that CeGRS1 effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 knock-out strain. Although both forms of CeGlyRS efficiently charged the cytoplasmic tRNAs Gly of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNA Gly isoacceptor, which carries a defective TC hairpin. Despite the WHEP domain per se lacking tRNA binding activity, deletion of this domain reduced the catalytic efficiency of the enzyme. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests a role for the WHEP domain as a regulator of the dynamic structure and activity of the enzyme. Aminoacyl-tRNA synthetases (aaRSs)3 belong to a ubiquitous and ancient family of enzymes that establish their genetic codes by attaching specific amino acids to their cognate tRNAs. The resultant aminoacyl-tRNAs are then delivered to ribosomes to decipher mRNA codons through base pairing with the anticodon of the aminoacyl-tRNA (1). Because protein translation takes place in both the cytoplasm and mitochondria in eukaryotes, two distinct sets of aaRSs are required: one functioning in the cytoplasm and the other functioning in mitochondria (1-4). In most cases, the cytoplasmic and mitochondrial forms of an aaRS are encoded by two different nuclear genes. Occasionally, both isoforms of an aaRS are encoded by the same nuclear gene through alternative initiation of translation, examples of which include genes encoding yeast alanyl-, glycyl-, histidyl-, and valyl-tRNA synthetases (5-9). As a result, cytoplasmic and mitochondrial forms of an aaRS, for example yeast glycyl-tRNA synthetase (GlyRS), possess essentially the same polypeptide sequence, except for a cleavable mitochondrial targeting signal (MTS) attached at the N terminus of the mitochondrial precursor form.GlyRS is one of the most intriguing aaRSs because of its divergent quaternary structure and evolutionary origin. Two distinct oligomeric structures of GlyRS exist: one with an ␣ 2 structure and the other with an ␣ 2 ␤ 2 structure (10, 11). These two forms are divergent not only in subunit composition but also in molecular size and protein sequence (12-14). Even so, they possess the same signature motifs and are thus assigned to the same class (class II). To date, ␣ 2 ␤ 2 enzymes exist only in bacteria and chloroplasts, but ␣ 2 enzymes are spread over all three domains of ...

No abstract