Elongacijski faktor Tu kao potencijalni marker mistranslacijskog stresa u bakteriji Escherichia coli

master's thesis

• Title (english): Elongation factor Tu as a potential marker of mistranslational stress in Escherichia coli
• Author: Andrea Hloušek-Kasun
• Mentor: Ita Gruić Sovulj (mentor)
• Committee member: Ita Gruić Sovulj (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-01-03, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Aminoacil-tRNA-sintetaze (aaRS) su enzimi koji kovalentno povezuju odgovarajuću molekulu tRNA s njoj pripadnom aminokiselinom u reakciji aminoaciliranja. Nastala aminoacilirana tRNA se pomoću elongacijskog faktora Tu (EF-Tu) doprema do ribosoma gdje sudjeluje u procesu biosinteze proteina. Ukoliko aaRS pogriješe pri odabiru pripadne aminokiseline ili tRNA, dolazi do ugradnje nepripadne aminokiseline u proteine (mistranslacije). Pokazano je da leucil-tRNA-sintetaza (LeuRS) iz bakterije Escherichia coli ponekad griješi pri diskriminaciji pripadnog leucina od norvalina, zbog čega je evolucijski razvila deacilacijsku domenu koja omogućuje popravak pogreške. Inaktiviranjem deacilacijske domene dolazi do povremene ugradnje norvalina u uvjetima njegove povišene koncentracije. Stanice s norvaliranim proteomom pokazuju značajno smanjenu vijabilnost, a smatra se da bi to mogla biti posljedica nepravilnog smatanja i agregacije mistranslatiranih proteina. Stoga je u ovom diplomskom radu, kako bi stekli uvid u stanični odgovor na mistranslacijski stres, odabran EF-Tu kao modelni protein. Dobiveni rezultati pokazuju da u uvjetima mistranslacijskog stresa, ipak ne dolazi do značajne promjene u njegovoj stabilnosti te povećane agregacije u citoplazmi, već smanjenja njegove razine. Stoga je EF-Tu zanimljiv modelni protein za izučavanje biološkog odgovora bakterijske stanice na mistranslaciju. Međutim, da bi se utvrdio utjecaj mistranslacije na strukturu i topivost proteina, potrebno je provesti dodatna istraživanja i biokemijske analize drugih modelnih proteina.

Abstract (english)

Aminoacyl-tRNA-synthetases (aaRS) are enzymes that covalently couple tRNA molecules with their cognate amino acids in aminoacylation reaction. The aminoacyl tRNAs are then escorted to the ribosome by the elongation factor Tu (EF-Tu) for protein biosynthesis. Deficiencies in aaRS editing mechanisms can lead to mistranslation and incorporation of noncognate amino acids into proteins. It has been shown that Escherichia coli LeuRS sometimes misacylates norvaline instead its cognate leucine. Consequently, through evolution LeuRS has developed a deacilation domain which ensures error correction. Deactivation of LeuRS deacylation domain leads to norvaline misincorporation in growth conditions where norvaline accumulates. Cells with high level of norvaline incorporation show loss in cell viability, probably because of misfolding and aggregation of mistranslated proteins. To obtain insights into biological cell response to mistranslational stress, we decided to monitor EF-Tu as a model protein. Our results show that mistranslated EF-Tu does not aggregate in bacterial cells nor does it exhibit changes in its stability. However, we observed that mistranslation leads to decrease in the EF-Tu levels. Hence, we concluded that EF-Tu could be an interesting model protein for investigation of biological cell response to mistranslation. In order to gain clear insights in misfolding and aggregation of proteins due to stress conditions caused by mistranslation, it is necessary to monitor and biochemically characterize other possible model proteins.

• Keywords: aminoacil-tRNA-sintetaze; elongacijski faktor Tu; mistranslacija; Escherichia coli; leucil-tRNA-sintetaza; norvalin; stanični odgovor; modelni protein
• Keywords (english): aminoacyl-tRNA-synthetases; elongation factor Tu; mistranslation; Escherichia coli; leucyl-tRNA synthetase; norvaline; cell response; model protein
• Language: croatian
• URN:NBN: urn:nbn:hr:217:575209
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-03-15 12:41:31