Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme

et al. (2014) Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme. PLoS ONE 9(4): e94659. doi:10.1371/journal.pone.0094659 Functional Substitution of a Eukaryotic Glycyl-tRNA Synthetase with an Evolutionarily Unrelated Bacterial Cognate Enzyme Chin-I Chien 0 Yu-Wei Chen 0 Yi-Hua Wu 0 Chih-Yao Chang 0 Tzu-Ling Wang 0 Chien-Chia Wang 0 Beata G. Vertessy, Institute of Enzymology of the Hungarian Academy of Science, Hungary 0 1 Department of Life Sciences, National Central University , Jung-li, Taiwan , 2 Department of Neurology, Landseed Hospital , Ping-jen, Taiwan , 3 Graduate Institute of Mathematics and Science Education, National Hsinchu University of Education , Hsinchu , Taiwan Two oligomeric types of glycyl-tRNA synthetase (GlyRS) are found in nature: a a2 type and a a2b2 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 a2-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1strain, while Escherichia coli GlyRS (a a2b2-type enzyme) and A. thaliana organellar GlyRS (a (ab)2-type enzyme) failed to rescue either defect of the yeast mull allele. However, a head-to-tail ab fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a a2-type eukaryotic GlyRS may be functionally substituted with a a2b2-type bacterial cognate enzyme despite their remote evolutionary relationships. - Funding: This work was supported in part by grants (NSC101-3113-B-008-001-MY3 and NSC102-2311-B-008-004-MY3 to CCW) from the National Science Council (Taipei, Taiwan) and in part by a grant (NCU-LSH-102-A-002) from the National Central University and Landseed Hospital Joint Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code by coupling amino acids specifically to their cognate tRNAs. The resultant aminoacyl-tRNA is then delivered by an elongation factor to ribosomes for protein synthesis. The anticodon of a tRNA reads the codon of mRNA by forming Watson-Crick base pairing. Prokaryotes contain a basic set of 1820 aaRs, but eukaryotes contain more, as they possess a second compartment (mitochondria) capable of protein translation [13]. For example, yeast possesses two distinct sets of aaRSs, one localized to the cytoplasm and the other to mitochondria. Each set is specific for cognate tRNAs within its respective cellular compartment, and it is sequestered from isoacceptors that are confined in other compartments. Cytoplasmic and mitochondrial isoforms of an aaRS with a given amino acid specificity are most often encoded by two distinct nuclear genes, regardless of the cellular compartments in which they are localized. However, four Saccharomyces cerevisiae genes, ALA1 (which encodes alanyl-tRNA synthetase) [4], GRS1 (which encodes glycyl-tRNA synthetase [GlyRS]) [5], HTS1 (which encodes histidyl-tRNA synthetase) [6], and VAS1 (which encodes valyl-tRNA synthetase) [7], specify both the cytoplasmic and mitochondrial functions. This dual-functional feature has been conserved in homologues of these genes in almost all yeast species studied [810]. In addition, the yeast GUS1 gene also specifies both the cytoplasmic and mitochondrial functions; its protein product acts as a glutamyl-tRNA synthetase in the cytoplasm and as a non-discriminating glutamyl-tRNA synthetase in mitochondria [11]. On the basis of conserved sequence motifs, the quaternary structure, and aminoacylation function, aaRSs can be divided into two classes of 10 enzymes each [12,13]. Class I enzymes possess two signature sequences, HIGH and KMSKS, while class II enzymes contain three degenerate motifs_motifs 1, 2, and 3. In addition, class I enzymes first couple amino acids to the 29-OH of the terminal adenylate residue of tRNA before transferring it to the 39-OH, while class II enzymes directly couple it to the 39-OH. Normally, orthologous enzymes that couple the same amino acid to isoaccepting tRNAs share convincing sequence similarities in their catalytic core domains and are grouped into the same class (I or II), suggesting that they descend from a common ancestor. However, three exceptions to this rule exist: lysyl-tRNA synthetase, GlyRS, and phenylalanyl-tRNA synthetase. (...truncated)