Utjecaj mistranslacije na stabilnost modelnih proteina u staničnom ekstraktu

master's thesis

• Title (english): Effect of mistranslation on the stability of model proteins in cell extract
• Author: Alojzije Brkić
• Mentor: Ita Gruić Sovulj (mentor)
• Mentor: Marko Močibob (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Ivica Đilović (član povjerenstva)
• Committee member: Snežana Miljanić (član povjerenstva)
• Committee member: Iva Juranović Cindrić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2019-02-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

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.

Abstract (english)

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.

• Keywords: izoleucil-tRNA-sintetaza; norvalin; mistranslacija; mistranslatirani proteini; temperatura mekšanja
• Keywords (english): isoleucyl-tRNA synthetase; norvaline; mistranslation; mistranslated proteins; melting point
• Language: croatian
• URN:NBN: urn:nbn:hr:217:995612
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-02-28 11:25:41