SVIBOR - Project code: 1-08-070

MINISTRY OF SCIENCE AND TECHNOLOGY

Strossmayerov trg 4, HR - 10000 ZAGREB
tel.: +385 1 459 44 44, fax: +385 1 459 44 69
E-mail: ured@znanost.hr

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Project code: 1-08-070


AMINOACYL-tRNA SYNTHETASES: GENE STRUCTURE AND PROTEIN FUNCTION


Main researcher: KUĆAN, ŽELJKO (24372)



Assistants
Type of research: basic
Duration from: 01/01/91. to 12/31/93.

Papers on project (total): 44
Papers on project quoted in Current Contents: 15
Institution name: Prirodoslovno-matematički fakultet, Prirodoslovni odjeli, Zagreb (119)
Department/Institute: Department of Organic Chemistry and Biochemistry
Address: Strossmayerov trg 14
City: 10000 - Zagreb, Croatia
Communication
Phone: 385 (01) 434-769, ¦. Ku†an
Fax: 385 (01) 432-526, ¦. Ku†an
Phone: 385 (01) 4561-197, I. Weygand
Fax: 385 (01) 425-497, I. Weygand
E-mail: Weygand@olimp.irb.hr

Summary: The goal of this project is to elucidate the principles underlying the highly specific interaction between tRNA and aminoacyl-tRNA synthetases, by employing the methods of biochemistry, molecular biology and genetics. The formation of the aminoacyl-tRNA is the crucial step for maintaining the fidelity of protein biosynthesis. We have focused our investigation on two tRNA/aminoacyl-tRNA synthetase systems from Saccharomyces cerevisiae: one specific for tyrosine and the other for serine. These two enzymes belong to two classes of aminoacyl-tRNA synthetases, which are characterized by different mode of interaction with their cognate tRNAs. Our experimental work deals with cloning, sequencing and the expression of the genes, in vivo and in vitro mutagenesis followed by selection of the mutants, and in some cases the determination of biochemical and biophysical parametars for tRNA/synthetase complex formation. In this way, we hope to identify the regions of the enzymes responsible for specific recognition of their cognate tRNA substrates and discrimination against non-cognate ones.

Keywords: protein biosynthesis, aminoacyl-tRNA synthetases, tRNA, mutagenesis, supressor tRNAs, gene expression

Research goals: The goal of our investigation is to get insight into highly specific intraction of yeast seryl- and tyrosyl-tRNA synthetases (SerRS and TyrRS, respectively) with their substrates: ATP,serine or tyrosine, and cognate tRNAs, and possibly to determine the function of the motifs not included in substrate recognition. Serine and tyrosine systems have been chosen for following reasons: (1) These enzymes belong to two classes of synthetases and different mode of interaction with their substrates is expected. (2) The crystal structures of both enzymes from prokarytic source are known, which provides the basis for interesting comparative study. (3) We are dealing with enzymes we have great experience with, starting from the first complete purification of tyrosyl-tRNA synthetase from a haploid yeast (Ž.Kućan&R.Chambers, J.Biochem.73(1973)811), to cloning of the SES1 gene, coding for seryl-tRNA synthetase (I.Weygand-Đurašević et al.,Nucleic Acids Res.15(1987)1887). Besides well established procedures for purification of specific tRNA species by conventional chromatographic columns, we are trying to construct various synthetic tRNA genes and transcribe them in vitro. This would allow the introduction of mutations into the most complex synthetase substrate, and the study of the importance of particular nucleotide for the accuracy of the aminoacylation.


COOPERATION - PROJECTS


  1. Name of project: PA-91-77 FIRCA Transfer RNA Recognition by Class II Synthetases
    Name of institution: Yale University
    City: 06511 - New Haven, CT, USA

  2. Name of project: GE/GLO/90/004 Protein engineering of aminoacyl-tRNA synthetases
    Name of institution: Faculty of Medicine , University of Chile
    City: 7 - Santiago, Chile


COOPERATION - INSTITUTIONS


  1. Name of institution: Institut de Biologie Moleculaire et Cellulaire C.N.R.S.
    Type of institution: University/Faculty
    Type of cooperation: Occasional exchange of experts
    City: 67084 - Strasbourg, Francuska

  2. Name of institution: EMBL Grenoble Outstation
    Type of institution: University/Faculty
    Type of cooperation: Occasional exchange of experts
    City: 38042 - Grenoble, Francuska

  3. Name of institution: Institut Ruđer Bošković
    Type of institution: University/Faculty
    Type of cooperation: Joint project
    City: 10000 - Zagreb, Croatia

  4. Name of institution: Institut fur Physiolgische Chemie, Universitat Bonn
    Type of institution: University/Faculty
    Type of cooperation: Systematic exchange of information
    City: Bonn, Njemačka

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Last update: 10/09/95
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