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Izoleucil-tRNA-sintetaza (IleRS) katalizira sintezu Ile-tRNAIle u procesu biosinteze proteina. IleRS može ponekad griješiti i misacilirati tRNAIle s valinom ili neproteinogenim norvalinom. Pogreška u biosintezi proteina (mistranslacija) može biti štetna za stanicu. Iz tog razloga, IleRS posjeduje domenu za popravak vlastite pogreške. U okviru ovog rada praćena je promjena temperature mekšanja β-galaktozidaze kao modelnog proteina uslijed zamjene izoleucina s valinom ili norvalinom. Termička stabilnost β-galaktozidaze praćena je u staničnim ekstraktima soja bakterije Escherichia coli koji ima inaktiviranu domenu za popravak pogreške IleRS te ugrađuje valin i norvalin na izoleucinske položaje. Pokazano je da mistranslacija izoleucinskih položaja β-galaktozidaze objema nepripadnim aminokiselinama smanjuje termičku stabilnost enzima te da je utjecaj norvalina izraženiji. Nadalje, opažena je značajnija inhibicija rasta bakterijskih stanica uslijed zamjene izoleucina norvalinom u odnosu na valin. Rezultati sugeriraju da veća toksičnost mistranslacije norvalinom barem djelomično potječe od smanjene stabilnosti proteina s ugrađenim norvalinom. Isoleucyl-tRNA synthetase (IleRS) catalyses formation of Ile-tRNAIle for protein biosynthesis. IleRS may mistakingly activate tRNAIle with valine and nonproteinogenic norvaline. However, IleRS uses an editing domain and corrects its own mistakes to prevent accumulation of erroneous proteins. This work focused on the effects of isoleucine to valine or isoleucine to norvaline substitutions on the thermal stability of a model protein, β-galactosidase. The melting point of β-galactosidase was measured in cell extracts of an IleRS editing-deficient strain of Escherichia coli, which incorporated valine or norvaline at isoleucine positions. We showed that mistranslation of β-galactosidase with both norvaline and valine lowered the thermal stability of the enzyme, with norvaline introducing a higher effect. Besides that, isoleucine to norvaline substitution inhibited the growth of the editing-deficient strain to a higher extent than isoleucine replacement with valine. The results suggest that higher toxicity of norvaline misincorporation may be in part caused by production of norvalylated proteins with a lower thermal stability. |
