| Popis: |
Mistranslation can occur due to inaccurate amino acid selection by aminoacyl-tRNA synthetases (aaRSs). Although usually toxic, due to the production of aberrant proteins, there are a few examples of its adaptive effect on cellular responses to subsequent stresses. Isoleucyl-tRNA synthetase (IleRS) is one of aaRSs that possess editing domain and hydrolyze incorrect aa-tRNA product. In our experiments, we used Escherichia coli strain expressing IleRS with inactivated editing domain, which allowed us to induce mistranslation by incubating bacteria in the medium supplemented with amino acids structurally similar to isoleucine. We simulated variable mistranslation rates using different concentrations (0.25, 0.5, 0.75 and 1 mM) of valine (Val) or norvaline (Nva). Mistranslation induced formation of elongated bacterial filaments, whose length correlated with increasing Val or Nva concentrations. Transmission electron microscopy showed morphological and ultrastructural changes in bacteria under stress, such as numerous intracellular vesicles and bulge formation. Survival assays revealed significantly increased survival of bacteria grown with 0.5, 0.75 and 1 mM Val or Nva in the presence of 1 mM H2O2. 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. |
