Cloning, overexpression and purification of Escherichia coli tRNAIle and tRNAVal

Cvetešić, Nevena (2010) Cloning, overexpression and purification of Escherichia coli tRNAIle and tRNAVal. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

Aminoacyl-tRNA synthetases (aaRS) are enzymes that covalently join amino acids with their cognate transfer RNAs (tRNAs). As highly accurate aminoacyl-tRNA synthesis is a prerequisite for the precise transmission of genetic information, some aaRSs have developed editing mechanisms to correct initial errors in amino acid activation (pre-transfer) or aminoacyltRNA formation (post-transfer). tRNA often stimulates pre-transfer editing by a yet unknown mechanism. In some cases, tRNA has to be posttranscriptionally modified (tRNAIle for example) in order to stimulate editing. Large amounts of biologically active tRNAs are needed to conduct detailed kinetic studies of tRNA-dependent editing by isoleucyl- and valyl-tRNA synthetases. Recombinant plasmids for in vivo overexpression of tRNAIle and tRNAVal under control of T7 strong promotor have been produced. The overexpression has been optimized for preparative purposes and an efficient purification method by reverse phase chromatography has been developed for purification of posttranscriptionally modified tRNAs. The properties of obtained tRNAs were assesed by enzyme kinetics.

Item Type: Thesis (Diploma thesis)
Keywords: tRNA, aminoacyl-tRNA synthetases, editing mechanisms, reverse phase chromatography, posttrancriptional modifications
Supervisor: Gruić Sovulj, Ita
Date: 2010
Number of Pages: 78
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 04 Sep 2014 11:37
Last Modified: 16 Feb 2016 13:57
URI: http://digre.pmf.unizg.hr/id/eprint/2630

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