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Translation is an essential cellular process in protein biosynthesis. Enzymes aminoacyl-tRNA synthetases (aaRS) have a key role as they attach tRNAs with their cognate amino acids. Aminoacylation is a two-step reaction consisting of: activation of amino acid utilizing ATP to form an aminoacyl-adenylate intermediate and transfer of aminoacyl moiety to cognate tRNA. Due to the structural and chemical similarity of some amino acids, a group of aaRSs developed post-transfer editing mechanism that facilitates hydrolysis of misaminoacylated tRNA to reduce mistranslation rate. Mistranslation has deleterious effects on a cell leading even to cell death, but in some cases beneficial effect of mistranslation was found in response to other stress conditions. To investigate effect of mistranslation on oxidative stress, Escherichia coli strain expressing mutant variant of isoleucyl-tRNA synthetase (IleRS) with inactivated editing domain was used in experiments. Variable mistranslation rates were induced by adding different concentrations (0.25, 0.5, 0.75 and 1 mM) of noncognate proteinogenic valine (Val) or nonproteinogenic norvaline (Nva) in the medium. Oxidative stress was induced by adding 1 mM H2O2. Light and transmission electron microscopy showed morphological changes, namely formation of filaments, as well as ultrastructural changes in bacteria under stress. Survival assays revealed a significant increase in the survival of bacteria in the presence of 1 mM H2O2 if they are grown with 0.5, 0.75 and 1 mM Val or Nva prior to induction of oxidative stress. Growth curve measurements showed that bacteria preincubated with Val and Nva grow better in oxidative stress conditions. Better growth with Val was concentration dependent, while 0.5 mM concentration had the most prominent effect for Nva. If bacteria were exposed simultaneously to both mistranslation and oxidative stress, higher mistranslation rates have shown to be beneficial as well, however, the effect was only observed during early exposure to oxidative stress. The results show that there is mistranslation-induced adaptation to oxidative stress. Further work will focus on proteome analysis in order to clarify cellular mechanisms that increase survival and growth under oxidative stress due to misincorporation of valine or norvaline at isoleucine positions in proteins. |
