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Site≠directed mutagenesis of tryptofan residues in the structure of the catalytic module of tyrosil≠tRNA synthetase Bos taurus
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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.
Key words: catalytic module of tyrosyl-tRNA synthetase, site-directed mutagenesis, cDNA, PCR amplification, DNA polymerase
E-mail: v.n.zayets gmail.com, a.tsuvariev gmail.com, babenko_lesia ukr.net, kornelyuk imbg.org.ua
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. 461Ц480. 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. 349Ц359. 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. 61Ц67.
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. 1597Ц1605. 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. 168Ц172. 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. 145Ц153. 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. 5762Ц5779.
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. 12Ц19. 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. 1363Ц1370. 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. 705Ц711. 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. 893Ц898.
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. 731Ц737. 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. 156Ц161.
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. 283Ц298. 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. 83Ц88. 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. 317Ц322.
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. 130Ц136. 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. 157Ц178. 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. 23Ц28. 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. 105Ц113. 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. 302Ц307. 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. 525Ц538. 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. 85Ц90.
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. 225Ц232.
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. 376Ц378.
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. 8091Ц8104. https://doi.org/10.1093/nar/gkx455
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