Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom

Marasović, Mirela

prikaz prve stranice dokumenta Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom

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master's thesis

Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom

Zagreb: University of Zagreb, Faculty of Science, 2011. urn:nbn:hr:217:026522

Marasović, Mirela

University of Zagreb
Faculty of Science
Department of Biology

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APA 6th Edition

Marasović, M. (2011). Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:026522

MLA 8th Edition

Marasović, Mirela. "Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom." Master's thesis, University of Zagreb, Faculty of Science, 2011. https://urn.nsk.hr/urn:nbn:hr:217:026522

Chicago 17th Edition

Harvard

Marasović, M. (2011). 'Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom', Master's thesis, University of Zagreb, Faculty of Science, accessed 18 April 2024, https://urn.nsk.hr/urn:nbn:hr:217:026522

Vancouver

Marasović M. Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2011 [cited 2024 April 18] Available at: https://urn.nsk.hr/urn:nbn:hr:217:026522

IEEE

M. Marasović, "Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2011. Available at: https://urn.nsk.hr/urn:nbn:hr:217:026522

Cite this item: https://urn.nsk.hr/urn:nbn:hr:217:026522

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Title	Podjedinica H arhejske RNA-polimeraze: od kloniranja do istraživanja interakcije s atipičnom seril-tRNA-sintetazom
Title (english)	Archaeal RNA polymerase subunit H: from cloning to investigation of interaction with atypical seryl-tRNA synthetase
Author	Mirela Marasović
Mentor	Ivana Weygand-Đurašević (mentor)
Committee member	Ivana Weygand-Đurašević (predsjednik povjerenstva)
Committee member	Željka Vidaković Cifrek (član povjerenstva)
Committee member	Ivana Ivančić Baće (član povjerenstva)
Granter	University of Zagreb Faculty of Science (Department of Biology) Zagreb
Defense date and country	2011, Croatia
Scientific / art field, discipline and subdiscipline	NATURAL SCIENCES Biology
Abstract	Interakcija podjedinice H arhejske RNA-polimeraze (RpoH) i seril-tRNA-sintetaze metanogenog tipa iz arheje Methanothermobacter thermautotrophicus (MtSerRS) detektirana je ranije u laboratoriju sustavom dvaju hibrida. Asocijacija MtSerRS:RpoH vrlo je zanimljiva budući da se radi o proteinima uključenim u proces koji je od vitalne važnosti za život stanice – ekspresiju gena. Funkcija arhejske RNA-polimeraze je transkripcija svih gena, dok MtSerRS aminoacilira tRNASer serinom te na taj način opskrbljuje ribosom supstratima za elongacijsku fazu translacije. U svrhu potvrde stvaranja kompleksa MtSerRS:RpoH tehnikama in vitro, bilo je potrebno pročistiti pojedine proteine u dovoljnim količinama. Zato je gen rpoH umnožen lančanom reakcijom polimeraze i ugrađen u vektor pod kontrolom jakog T7 promotora. Optimiran je sustav za prekomjernu ekspresiju i pročišćavanje RpoH iz bakterije E.coli, a zatim je RpoH pročišćen u preparativnoj količini. Također, prekomjerno je eksprimirana i pročišćena MtSerRS. Interakcija dvaju proteina istražena je gel-retardacijskom elektroforezom i supročišćavanjem kompleksa na koloni. Preliminarni rezultati dali su naznaku stvaranja kompleksa na nativnom gelu, međutim supročišćavanjem kompleksa na koloni asocijacija MtSerRS:RpoH nije detektirana.
Abstract (english)	A yeast two hybrid screen performed earlier in our laboratory revealed interaction of Archaeal RNA polymerase subunit H (RpoH) and methanogenic-type seryl-tRNA synthetase from Archaea Methanothermobacter thermautotrophicus (MtSerRS). Association MtSerRS:RpoH is very interesting since both proteins are involved in a process vital for cell's life – expression of genes. Archaeal RNA polymerase transcribes genes while MtSerRS aminoacylates tRNASer with serine and supplies ribosome with substrates for elongation phase of translation. In order to confirm the formation of MtSerRS:RpoH complex by in vitro methods, it was nessesary to obtain the individual proteins in sufficient amounts. Therefore, rpoH gene was amplified using PCR and cloned in a vector under control of T7 strong promoter. The system for overexpression and purification of RpoH in Escherichia coli was oprimized, and then RpoH was purified for preparative purposes. Also, MtSerRS was overexpressed and purified. The interaction between two proteins was investigated by gel-retardation electrophoresis and pull-down assay. Preliminary results indicate complex formation on native gel, but the association MtSerRS:RpoH was not detected by pull-down assay.
Keywords
Keywords (english)
Language	croatian
URN:NBN	urn:nbn:hr:217:026522
Study programme	Title: Biology; specializations in: Molecular biology Course: Molecular biology Study programme type: university Study level: graduate Academic / professional title: magistar/magistra biologije (magistar/magistra biologije)
Type of resource	Text
File origin	Born digital
Access conditions	Open access
Terms of use
Repository	Repository of the Faculty of Science
Created on	2018-04-09 08:44:44

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