Uvođenje mutacija u sintetsko mjesto izoleucil-tRNA-sintetaze s ciljem rasvjetljavanja mehanizama hidrolitičkog popravka

master's thesis

• Title (english): Towards elucidation of hydrolitic proofreading mehanism by mutational analysis of isoleucil-tRNA-synthease synhetic site
• Author: Nina Franičević
• Mentor: Ita Gruić Sovulj (mentor)
• Committee member: Ita Gruić Sovulj (predsjednik povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Dubravka Hranilović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2011-07-04, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Aminoacil-tRNA-sintetaze (aaRS) su enzimi koji kataliziraju reakciju kovalentnog vezanja aminokiseline na odgovarajuću molekulu tRNA. Uslijed nemogućnosti izbora pripadne aminokiseline s visokim stupnjem točnosti, neke aaRS su razvile mehanizme kojima popravljaju pogreške pri aminoacilaciji. Popravkom prije prijenosa uklanjaju se pogreške nastale prilikom aktivacije nepripadne aminokiseline, a popravkom poslije prijenosa one nastale njenim prijenosom na tRNA. Najnovija istraživanja upućuju na to da se kod izoleucil-tRNA-sintetaze (lleRS) popravak prije prijenosa odvija u sintetskom mjestu enzima, gdje dolazi do kinetičke razdiobe nepripadnog aminoacil-adenilata između reakcija hidrolize i prijenosa aminokiseline na tRNA. Ciljana mutageneza odabranih bočnih ogranaka u sintetskom mjestu i kinetička karakterizacija dobivenih mutanata IleRS pridonijele su razumijevanju mehanizma kojim se odvija reakcija aktivacije pripadne i nepripadne aminokiseline te hidroliza nepripadnih aminoacil-adenilata. Tako, hidrolitički popravak u sintetskom mjestu ovisi o preciznoj konformaciji aktivnog mjesta koja pozicionira nepripadni aminoacil-adenilat u hidrolitičko podmjesto. Dobiveni rezultati upućuju da Gly56, Tyr59 i Glu561 imaju važnu ulogu prilikom razlikovanja pripadnih i nepripadnih supstrata te pozicioniranja katalitičke molekule vode.

Abstract (english)

Aminoacyl-tRNA synthetases (aaRSs) catalyze covalent attachment of amino acids to their cognate tRNAs. Due to inherent inability of some aaRS to recognize cognate amino acid with a high accuracy, these aaRS have developed hydrolytic editing mechanisms to correct mistakes made during aminoacylation. Pre-transfer editing clears the noncognate aminoacyl-adenylates, while misacylated tRNAs are corrected by the post-transfer editing. Recent data show that pre-transfer editing by isoleucyl-tRNA synthetase (IleRS) occurs within the synthetic site, where noncognate aminoacyl-adenylate is kinetically partitioned between hydrolysis and the transfer step. Site-directed mutagenesis and kinetic characterization of the obtained IleRS synthetic site mutants contributed to understanding of the mechanisms of amino acid activation and noncognate aminoacyl-adenylate hydrolysis. Thus, pre-transfer editing depends on precise conformation of the synthetic site that positions the noncognate aminoacyl-adenylate in the hydrolytic subsite. We propose that Gly56, Tyr59 and Glu561 have important role in discrimination of the cognate and noncognate substrates and in positioning of the catalytic water molecule.

• Keywords: aminoacil-tRNA-sintetaze; mehanizmi popravka pogreške; popravak pogreške prije prijenosa; ciljana mutageneza; izoleucil-tRNA-sintetaza; hidroliza aminoacil-adenilata
• Keywords (english): aminoacyl-tRNA synthetases; editing mechanisms; pre-transfer editing; site-directed mutagenesis; isoleucyl-tRNA synthetase; hydrolysis of aminoacyl-adenylates
• Language: croatian
• URN:NBN: urn:nbn:hr:217:225583
• 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: Access restricted to students and staff of home institution
• Created on: 2017-05-03 08:17:52