dc.description.abstract | The yeast Saccharomyces cerevisiae possesses two GluRS enzymes that are encoded by two different nuclear genes. Cytoplasmic GluRS (GluRSc) is encoded by GUS1, while mitochondrial GluRS (GluRSm) is encoded by MSE1. GluRSc attaches glutamate to cytoplasmic tRNAnGlu and mitochondrial tRNAmGln. In contrast, GluRSm glutamylates only mitochondrial tRNAmGlu. As GluRSc can attach glutamate to both tRNAnGlu and tRNAmGln, it is called non-discriminating GluRS (ND-GluRS). On the other hand, GluRSm only recognizes tRNAmGlu, it is called discriminating GluRS (D-GluRS).In our study, we found that ND-GluRS of bacteria can rescue a yeast GluRSm knockout strain, but D-GluRS of eukaryotes and other bacteria cannot. Alignment of these GluRSs sequences showed that the amino acids responsible for tRNAGlu recognition are well conserved in yeast GluRSm and the aforementioned ND-GluRSs of bacteria. Comparison of the secondary structure of cytoplasmic tRNAnGlu, mitochondrial tRNAmGlu, and mitochondrial tRNAmGln showed that the first base pair in the accepter stem and nucleosides 33 and 36 in the anticodon loop are highly diverged and may serve as their identity elements. Unfortunately, in vitro transcribed tRNAmGlu and tRNAmGln were inactive for aminoacylation, and further mapping of identity elements in tRNA were thus impossible. Phylogenetic analysis showed that the GluRSm sequences of eukaryotes are clustered in a monophyletic branch that exhibits a higher affinity with GluRS of bacteria than with GluRS of archaea, suggesting that mitochondrial GluRSs of eukaryotes are descended from GluRSs of bacteria.
A second part of the thesis is focused on yeast glycyl-tRNA synthetase (GlyRS). In all yeast species, Saccharomyces cerevisiae and Vanderwaltozyma polyspora are the only two yeasts known to contain two GlyRS genes. GRS1 encodes both cytoplasmic and mitochondrial activities, whereas GRS2 is silent and dispensable under normal growth conditions. We show here that VpGlyRS2 (encoded by V. polyspora GRS2) effectively rescued the cytoplasmic defect of a S. cerevisiae GRS1 knockout strain when expressed from a constitutive promoter. Although VpGRS2 was practically silent under normal conditions, but its expression could be induced by high temperature, high pH, and ethanol. In addition, VpGlyRS2 was exclusively localized in the cytoplasm, more stable under heat-shock conditions in vivo, and almost as active as VpGlyRS1 in glycylation of tRNA in vitro. Our study reinforces the hypothesis that GRS2 of V. polyspora is an inducible gene that acts in response to stress to maintain the homeostasis of yeast cells.
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