Biopolym. Cell. 2006; 22(4):283-289.
Structure and Function of Biopolymers
Investigation of the interaction between isolated C-module of tyrosyl-tRNA synthetase and tRNA by fluorescence spectroscopy
- Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
Abstract
The non-catalytic COOH-terminal module of mammalian tyrosyl-tRNA synthetase manifests a dual function: involvement in tRNA binding as a cis-factor and cytokine activity after proteolytic cleavage from synthetase catalytic core similar to EMAP II cytokine. The C-module of TyrRS contains a single Trp144 which is an intrinsic fluorescent probe in protein structure, but it is localized outside of RNA binding site. To explore the interaction between C-module and tRNA in solution the conservative aromatic Phe127 residue was substituted for Trp127 fluorophore by site-directed mutagenesis. This replacement allowed enhancing the protein quantum yield and determining the binding parameters of tRNA and C-module. The dissociation constant of the complex between C-module and tRNAPhe was calculated on the basis of spectrofluorometric titrations data. It was about 2.9·10–8M, and stoichiometry of the complex n=1.2.
Keywords: tyrosyl-tRNA synthetase, mutagenesis, fluorescence
Full text: (PDF, in Ukrainian)
References
[1]
Mirande M. Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol. 1991;40:95-142.
[2]
Bonnefond L, Gieg? R, Rudinger-Thirion J. Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems. Biochimie. 2005;87(9-10):873-83.
[3]
Kornelyuk AI. Structural and functional investigation of mammalian tyrosyl-tRNA synthetase. Biopolym Cell. 1998; 14(4):349-59.
[4]
Kornelyuk AI, Tas MPR, Dubrovsky AL, Murray JC. Cytokine activity of the non-catalytic EMAP-2-like domain of mammalian tyrosyl-tRNA synthetase. Biopolym. Cell. 1999; 15(2):168-72.
[5]
Wakasugi K, Schimmel P. Two distinct cytokines released from a human aminoacyl-tRNA synthetase. Science. 1999;284(5411):147-51.
[6]
Quevillon S, Agou F, Robinson JC, Mirande M. The p43 component of the mammalian multi-synthetase complex is likely to be the precursor of the endothelial monocyte-activating polypeptide II cytokine. J Biol Chem. 1997;272(51):32573-9.
[7]
Simos G, Segref A, Fasiolo F, Hellmuth K, Shevchenko A, Mann M, Hurt EC. The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases. EMBO J. 1996;15(19):5437-48.
[8]
Kushiro T, Schimmel P. Trbp111 selectively binds a noncovalently assembled tRNA-like structure. Proc Natl Acad Sci U S A. 2002;99(26):16631-5.
[9]
Shalak V, Kaminska M, Mitnacht-Kraus R, Vandenabeele P, Clauss M, Mirande M. The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component. J Biol Chem. 2001;276(26):23769-76.
[10]
Murzin AG. OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J. 1993;12(3):861-7.
[11]
Moras D. Structural aspects and evolutionary implications of the recognition between tRNAs and aminoacyl-tRNA synthetases. Biochimie. 1993;75(8):651-7.
[12]
Kordysh MA, Odynets KA, Kornelyuk AI. Trp144 as a fluorescence probe for investigation of the C-module rapid conformation dynamics in eukaryotic tyrosyle-tRNA synthetase. Biopolym Cell. 2003; 19(5):436-9.
[13]
Kordysh MA, Kornelyuk AI. The monitoring of conformational changes of the Trp144 residue environment in C-module of tyrosyl-tRNA synthetase undr heat denaturation. Dopovidi Nats Akad Nauk Ukrainy. 2004; (1):156-61.
[14]
Kordysh MA, Kornelyuk AI. Fluorescence and dynamics of structural environment of TRP125 fluorophore in EMAP II cytokine. Biofiz Vistn. 2003; Iss.(13): 38-41.
[15]
Kordysh MA, Dubrovsky OL, Kornelyuk AI. Local conformational transition of Trp125 in EMAP II cytokine inducted by physiological temperature. Physics of the Alive. 2005; 13(1):79–85.
[16]
Dubrovsky AL, Brown Jn, Kornelyuk AI, Murray JC, Matsuka GKh. Bacterial expression of full-length and truncated forms of cytokine EMAP-2 and cytokine-like domain of mammalian tyrosyl-tRNA synthetase. Biopolym Cell. 2000; 16(3):229-235.
[17]
Kanibolotskiy DS, Odynets KA, Skurskiy SI, Kornelyuk AI. Study of intramolecular mobility of cytokine-like C-terminal module of tyrosyl-mammalian tRNA synthetase by molecular dynamics. Physics live. 2003; 11(2):61-71.
[18]
Roy S. Fluorescence quenching methods to study protein-nucleic acid interactions. Methods Enzymol. 2004;379:175-87.
[19]
Golub A, Petrushenko Z, Odynets K, Dubrovsky A, Rozhko O, Matsuka G, Solecka K, Olszak K, Przykorska A, Kornelyuk A. Cytokine-like C-terminal module of mammalian tyrosyl-tRNA synthetase reveals structure-specific tRNA bind ing: Computational docking modeling and footprint analysis. Aminoacyl-tRNA synthetases in biology, medicine, and evoluÂtion. Asilomar, 2002: 116.
[20]
Renault L, Kerjan P, Pasqualato S, M?n?trey J, Robinson JC, Kawaguchi S, Vassylyev DG, Yokoyama S, Mirande M, Cherfils J. Structure of the EMAPII domain of human aminoacyl-tRNA synthetase complex reveals evolutionary dimer mimicry. EMBO J. 2001;20(3):570-8.
[21]
Lakowicz J. R. Principles of fluorescent spectroscopy. 2nd Edition. New York: Plenum Press, 1999. 725 p.
[22]
Reshetnyak YK, Koshevnik Y, Burstein EA. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues. Biophys J. 2001;81(3):1735-58.
