AMINOACYL-tRNA SYNTHETASES: GENE STRUCTURE AND PROTEIN FUNCTION
Main researcher
: KUĆAN, ŽELJKO (24372) Assistants
NARANĐA, TATJANA (109286)
PODRAVEC, MARIJA (37751)
RUBELJ, IVICA (108443)
WEYGAND-ĐURAŠEVIĆ, IVANA (57696)
LANDEKA, IRENA (900209)
GRUIĆ, ITA (900661)
ROKOV, JASMINA (900834)
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.
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
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
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
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
Name of institution
: EMBL Grenoble Outstation Type of institution: University/Faculty Type of cooperation: Occasional exchange of experts City: 38042 - Grenoble, Francuska
Name of institution
: Institut Ruđer Bošković Type of institution: University/Faculty Type of cooperation: Joint project City: 10000 - Zagreb, Croatia
Name of institution
: Institut fur Physiolgische Chemie,
Universitat Bonn Type of institution: University/Faculty Type of cooperation: Systematic exchange of information City: Bonn, Njemačka Other information about the project.