Cytology and Genetics

, Volume 53, Issue 3, pp 219–226 | Cite as

Site-Directed Mutagenesis of Tryptophan Residues in the Structure of the Catalytic Module of Tyrosyl-tRNA Synthetase from Bos taurus

  • V. N. ZayetsEmail author
  • A. Yu. Tsuvarev
  • L. A. Kolomiiets
  • A. I. Kornelyuk


Site-directed mutagenesis of the N-terminal catalytic module of Bos taurus tyrosyl-tRNA synthetase (mini-BtTyrRS) with the substitution of three Trp residues by Ala residues in its structure using the modified QuikChange method was performed to study structural dynamic and functional properties of the protein by fluorescence spectroscopy. Point substitutions of tryptophan codons TGG with alanine codons GCG in the cDNA nucleotide sequence of the tyrosyl-tRNA synthetase catalytic module cloned in expression plasmid pET-30a were obtained during sequential PCR reactions using the developed primers. As a result, mini-BtTyrRS cDNAs, whose sequences contain only one tryptophan codon in each of the three positions in the protein structure, were obtained.


catalytic module of tyrosyl-tRNA synthetase site-directed mutagenesis PCR amplification DNA polymerase 



  1. 1.
    Pang, Y.L.J., Poruri, K., and Martinis, S.A., tRNA synthetase: tRNA aminoacylation and beyond, WIREs RNA, 2014, vol. 5, no. 4, pp. 461–480. CrossRefGoogle Scholar
  2. 2.
    Kornelyuk, A.I., Structural and functional investigation of mammalian tyrosyl-tRNA synthetase, Biopolym. Cell, 1998, vol. 14, no. 4, pp. 349–359. CrossRefGoogle Scholar
  3. 3.
    Gnatenko, D.V., Kornelyuk, A.I., Kurochkin, I.V., Ribkinska, T.A., and Matsuka, G.Kh., Isolation and characteristics of functionally active proteolytically modified form of tyrosyl-tRNA synthetase from the bovine liver, Ukr. Biochim. J., 1991, vol. 63, no. 4, pp. 61–67.Google Scholar
  4. 4.
    Greenberg, Y., King, M., Kiosses, W.B., Ewalt, K., Yang, X., Schimmel, P., Reader, J.S., and Tzima, E., The novel fragment of tyrosyl-tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells, FASEB J., 2008, vol. 22, no. 5, pp. 1597–1605. CrossRefGoogle Scholar
  5. 5.
    Kornelyuk, A.I., Maarten, P.R., Dubrovsky, A.L., and Murray, J.C., Cytokine activity of the non-catalytic EMAP-2-like domain of mammalian tyrosyl-tRNA synthetase, Biopolym. Cell, 1999, vol. 15, no. 2, pp. 168–172. CrossRefGoogle Scholar
  6. 6.
    Guo, M. and Schimmel, P., Essential non-translational functions of tRNA synthetases, Nat. Chem. Biol., 2013, vol. 9, pp. 145–153. CrossRefGoogle Scholar
  7. 7.
    Ladokhin, A.S., Fluorescence spectroscopy in peptide and protein analysis, in Meyers, R.A., Ed., Chichester: John Wiley and Sons Ltd., 2002, pp. 5762–5779.Google Scholar
  8. 8.
    Chatttopadhyay, A. and Haldar, S., Dynamic insight into protein structure utilizing red edge excitation shift, Acc. Chem. Res., 2013, vol. 47, no. 1, pp. 12–19. CrossRefGoogle Scholar
  9. 9.
    Rochamare, S.B. and Gaikwad, M., Tryptophan environment and functional characterization of a kinetically stable serine protease containing a polyproline II fold, J. Fluoresc., 2014, vol. 24, pp. 1363–1370. CrossRefGoogle Scholar
  10. 10.
    Kordysh, M. and Kornelyuk, A., Conformational flexibility of cytokine-like C-module of tyrosyl-tRNA synthetase monitored by Trp144 intrinsic fluorescence, J. Fluoresc., 2006, vol. 16, pp. 705–711. CrossRefGoogle Scholar
  11. 11.
    Turoverov, K.K. and Kuznetsova, I.M., The intrinsic fluorescence of globular actin: peculiarities in the location of tryptophan residues, Bioorg. Chem., 1998, vol. 24, no. 12, pp. 893–898.Google Scholar
  12. 12.
    Vallee-Belisle, A. and Michnick, S.W., Visualizing transient protein-folding intermediates by tryptophan-scanning mutagenesis, Nat. Struct. Mol. Biol., 2012, vol. 19, no. 7, pp. 731–737. CrossRefGoogle Scholar
  13. 13.
    Kordysh, M.A. and Kornelyuk, A.I., Monitoring of the conformational change in the environment of the Trp144 fluorophore in the C-module of tyrosyltRNA synthetase during thermal denaturation, Dop. Nac. Acad. Nauk Ukraine, 2004, no. 1, pp. 156–161.Google Scholar
  14. 14.
    Kordysh, M.A. and Kornelyuk, A.I., Investigation of the interaction between isolated C-module of tyrosyl-tRNA synthetase and tRNA by fluorescence spectroscopy, Biopolym. Cell, 2006, vol. 22, no. 4, pp. 283–298. CrossRefGoogle Scholar
  15. 15.
    Klimenko, I.V., Gushcha, T.O., and Kornelyuk, A.I., Properties of tryptophan fluorescence of two forms of tyrosyl-tRNA synthetase from the liver, Biopolym. Cell, 1991, vol. 7, no. 6, pp. 83–88. CrossRefGoogle Scholar
  16. 16.
    Kornelyuk, A.I., Klimenko, I.V., and Odynets, K.A., Conformational change of mammalian tyrosyl-tRNA synthetase induced by tyrosyladenylate formation, Biochem. Mol. Biol. Int., 1995, vol. 35, no. 2, pp. 317–322.Google Scholar
  17. 17.
    Kordysh M.O., Kyryushko G.V., Mely, Y., and Kornelyuk O.I. Conformational mobility investigation of TyrRS N-module and its complex with tRNA using the methods of time-resolved fluorescence spectroscopy, Biopolym. Cell, 2007, vol. 23, no. 2, pp. 130–136.
  18. 18.
    Ling, M.M. and Robinson, B.H., Approaches to DNA mutagenesis: an overview, Anal. Biochem., 1997, vol. 254, pp. 157–178. CrossRefGoogle Scholar
  19. 19.
    Inoue, H., Nojima, H., and Okayama, H., High efficiency transformation of Escherichia coli plasmids, Gene, 1990, vol. 96, pp. 23–28. CrossRefGoogle Scholar
  20. 20.
    Miller, E.M. and Nickoloff, J.A., Escherichia coli electrotransformation, Methods Mol. Biol., 1995, vol. 47, pp. 105–113. Google Scholar
  21. 21.
    Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989.Google Scholar
  22. 22.
    Morrison, K.L. and Weiss, G.A., Combinatorial alanine scanning, Curr. Opin. Chem. Biol., 2001, vol. 5, pp. 302–307. CrossRefGoogle Scholar
  23. 23.
    Liu, H. and Naismith, J.H., An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol, BMC Biotechnol., 2008, vol. 8, no. 1.
  24. 24.
    Vovis, G.F. and Lacks, S., Complementary action of restriction enzymes endo R-DpnI and endo R-DpnII on bacteriophage fI DNA, J. Mol. Biol., 1977, vol. 115, no. 3, pp. 525–538. (77)90169-3
  25. 25.
    Edelheit, O., Hanukoglu, A., and Hanukoglu, I., Simple and efficient site-directed mutagenesis using two single-primer reaction in parallel to generate mutants for protein structure-function studies, BMC Biotechnol., 2009, vol. 9, no. 1.
  26. 26.
    Qui, D. and Scholthof, R.-B.G., A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis, J. Virol. Methods, 2008, vol. 149, no. 1, pp. 85–90.CrossRefGoogle Scholar
  27. 27.
    Salerno, J.C., Jones, R.J., and Erdogan, E., A single-stage polymerase-based protocol for the introduction of deletions and insertion without subcloning, Mol. Biotechnol., 2005, vol. 29, no. 3, pp. 225–232.CrossRefGoogle Scholar
  28. 28.
    Tregan, A., Kielbus, M., Czapinski, J., Stepulak, A., Huhtaniemi, I., and Rivero-Muller, A., REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering, Sci. Rep., 2016, vol. 6.
  29. 29.
    Tseng, W.-Chi., Lin, J.-W., Wei, T.-Yu., and Fang, T.-Yu., A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method, Anal. Biochem., 2008, vol. 375, no. 2, pp. 376–378.CrossRefGoogle Scholar
  30. 30.
    Zheng, L., Bauman, U., and Reymnd, J.-L., An efficient one-step site-directed and site-saturation mutagenesis protocol, Nucleic Acids Res., 2004, vol. 32, no. 14. e115. CrossRefGoogle Scholar
  31. 31.
    Blocquel, D., Li Sh, Wei N., Daub H., Sajish M., Erfurth M.-L., Kooi G., Zhou J., Bai G., Schimmel P., Jordanova A., and Yang X.-L. Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy, Nucleic Acids Res., 2017, vol. 45, no. 13, pp. 8091–8104. CrossRefGoogle Scholar

Copyright information

© Allerton Press, Inc. 2019

Authors and Affiliations

  • V. N. Zayets
    • 1
    Email author
  • A. Yu. Tsuvarev
    • 1
    • 2
  • L. A. Kolomiiets
    • 1
  • A. I. Kornelyuk
    • 1
  1. 1.Institute of Molecular Biology and Genetics, National Academy of Sciences of UkraineKyivUkraine
  2. 2.Taras Shevchenko National University of KyivKyivUkraine

Personalised recommendations