TSitologiya i Genetika 2019, vol. 53, no. 3, 47-57
Cytology and Genetics 2019, vol. 53, no. 3, 219–226, doi: https://www.doi.org/10.3103/S009545271903006X

Site­directed mutagenesis of tryptofan residues in the structure of the catalytic module of tyrosil­tRNA synthetase Bos taurus

Zayets V.N., Tsuvarev A.Yu., Kolomiiets L.A., Kornelyuk A.I.

  1. Институт молекулярной биологии и генетики НАН Украины, Киев
  2. Киевский национальный университет имени Тараса Шевченка, Киев

SUMMARY. To study the structural-dynamic and functional properties of the N-terminal catalytic module of Bos taurus tyrosyl-tRNA synthetase (mini BtTyrRS) by fluorescence spectroscopy site-directed mutagenesis of the protein with the replacement of three Trp residues with Ala residues in its structure was performed using the modified QuikChang method. In the process of sequential PCR reactions using the developed primers point substitutions of tryptophan codons TGG with alanine codons GCG were made within the cDNA sequence of the tyrosyl-tRNA synthetase catalytic module cloned in the expression plasmid pET-30a. As a result, mini BtTyrRS cDNAs were obtained within the nucleotide sequence of which there is only one codon of tryptophan in each of the three positions in the protein structure.

Keywords: catalytic module of tyrosyl-tRNA synthetase, site-directed mutagenesis, cDNA, PCR amplification, DNA polymerase

TSitologiya i Genetika
2019, vol. 53, no. 3, 47-57

Current Issue
Cytology and Genetics
2019, vol. 53, no. 3, 219–226,
doi: 10.3103/S009545271903006X

Full text and supplemented materials

Free full text: PDF  


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. https://doi.org/10.1002/wrna.1224

2. Kornelyuk, A.I., Structural and functional investigation of mammalian tyrosyl-tRNA synthetase, Biopolym. Cell, 1998, vol. 14, no. 4, pp. 349–359. https://doi.org/10.7124/bc.0004DF

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.

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. https://doi.org/10.1096/fj.07-9973com

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. https://doi.org/10.7124/bc.000516

6. Guo, M. and Schimmel, P., Essential non-translational functions of tRNA synthetases, Nat. Chem. Biol., 2013, vol. 9, pp. 145–153. https://doi.org/10.1038/nchembio.1158

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.

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. https://doi.org/10.1021/ar400006z

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. https://doi.org/10.1007/s10895-014-1445-5

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. https://doi.org/10.1007/s10895-006-0113-9

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.

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. https://doi.org/10.1038/nsmb.2322

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.

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. https://doi.org/10.7124/bc.00073B

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. https://doi.org/10.7124/bc.000303

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.

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. https://doi.org/10.7124/bc.00075F

18. Ling, M.M. and Robinson, B.H., Approaches to DNA mutagenesis: an overview, Anal. Biochem., 1997, vol. 254, pp. 157–178. https://doi.org/10.1006/abio.1997.2428

19. Inoue, H., Nojima, H., and Okayama, H., High efficiency transformation of Escherichia coli plasmids, Gene, 1990, vol. 96, pp. 23–28. https://doi.org/10.1016/0378-1119(90)90336-P

20. Miller, E.M. and Nickoloff, J.A., Escherichia coli electrotransformation, Methods Mol. Biol., 1995, vol. 47, pp. 105–113. https://doi.org/10.1385/0-89603-310-4:105

21. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989.

22. Morrison, K.L. and Weiss, G.A., Combinatorial alanine scanning, Curr. Opin. Chem. Biol., 2001, vol. 5, pp. 302–307. https://doi.org/10.1016/S1367-5931(00)00206-4

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. https://doi.org/10.1186/1472-6750-8-91

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. doi.org/ (77)90169-3 https://doi.org/10.1016/0022-2836

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. https://doi.org/10.1186/1472-6750-9-61

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.

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.

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. https://doi.org/10.1038/srep19121

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.

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. https://doi.org/10.1093/nar/gnh110

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. https://doi.org/10.1093/nar/gkx455