engleski

Cellular responses to proteome-wide isoleucine mistranslation

Aminoacyl-tRNA synthetases (aaRS) attach amino acids onto cognate tRNAs. Aminoacylated tRNA is delivered to the ribosome where insertion of genetically encoded amino acid at the corresponding place in the proteome takes place. Some aaRSs, like isoleucyl-tRNA synthetase (IleRS), developed hydrolytic editing mechanisms to prevent mispairing of amino acid and tRNA, hence ensuring translational fidelity. Naturally occurring amino acids valine (Val) and norvaline (Nva) are equally good non-cognate substrates for IleRS and therefore represent a threat to faithful protein synthesis in Escherichia coli. Inactivation of the IleRS editing mechanisms makes the enzyme error-prone and leads to more frequent incorporation of Nva or Val instead of isoleucine (Ile) in the proteome (mistranslation). Intriguingly, substitution of Ile with Nva appears to be more toxic than Ile- to-Val replacement. Mistranslation affects protein folding resulting in growth defects and neurogenerative diseases in mammals. We investigated how E. coli tunes the apparatus for maintaining proteostasis to minimize the damage related to mistranslation. Two different types of substitutions were explored: canonical (Ile-to-Val) and noncanonical (Ile-to-Nva). Cellular responses to mistranslation were investigated by quantitative proteomics to reveal changes both in the proteome and DnaK clients isolated in pull-down assay. DnaK is the central chaperone in E. coli and therefore, chosen to be a model chaperone in the performed experiments. To induce mistranslation in E. coli we constructed a strain with editing deficient IleRS and mediated its growth in the media supplemented with Nva or Val (c = 1 mM). Under such conditions, E. coli exhibited growth defect and displayed 10 % (Ile-to-Nva) or 16 % (Ile-to-Val) mistranslation frequencies in its proteins. Further proteome analysis revealed significant upregulation of chaperones, among them DnaK and GroES/EL – both part of two main chaperone systems in E. coli. However, a protease response seemed to remain unaltered. Intriguingly, mistranslation did not change the identity of DnaK clients and no noticeable difference between cellular responses to canonical and noncanonical mistranslation was observed. Our data support the notion that proteome-wide substitution of Ile with either Nva or Val caused growth defect and provoked chaperone response that helps E. coli to survive at 37 °C. In search for conditions where mistranslation could be more damaging, we tested the growth of the mistranslating strain under error-prone conditions at 42 °C. The lack of growth under these conditions demonstrated that combination of heat-shock and mistranslation highly effects E coli. This may originate from the overload of the cell apparatus for maintaining proteostasis and is yet to be investigated.

isoleucyl-tRNA synthetase ; mistranslation ; DnaK ; valine ; norvaline

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