CLONING OF CDNA-GENE OF ARABIDOPSIS THALIANA RIBOSOMAL PROTEIN S6, ITS EXPRESSION IN ESCHERICHIA COLI AND ISOLATION OF ATRPS6A1 RECOMBINANT PROTEIN
DOI:
https://doi.org/10.26577/eb.2022.v91.i2.012Keywords:
phosphorylation, cloning, recombinant protein, plant ribosomal protein S6 (pRPS6)Abstract
Ribosomal protein S6 (RPS6) is a component of eukaryotic 40S ribosomal subunits (40S-RSu) that regulates the translation of certain types of mRNA. RPS6 is the unique 40S-RSu protein which is able to phosphorylation, it is one of the targets for the regulation of protein biosynthesis in eukaryotes. There is evidence that ribosomes with the highest level of phosphorylation of RPS6 create selective advantages in translation of mRNAs encoding a lot of the protein components of the cell translational apparatus. Understanding the mechanism of regulation of plant biosynthesis through phosphorylation of pRPS6 will increase plant biomass and yield.
In this work, the AtRPS6A1 cDNA gene encoding the ribosomal protein S6, was cloned into the pET19b vector. This gene is expressed in Escherichia coli cells, and the recombinant PRS6 protein encoded by it is isolated by ion and affinity (IMAC) chromatography, purified by dialysis and concentrated. The PRS6 protein will be used in in vitro experiments to study the molecular mechanisms of regulation of plant mRNA translation through its phosphorylation and production of monoclonal antibodies to this protein.
References
Borlaug N. Feeding a hungry world // Science. – 2007. – Vol. 318. – P. 359-359.
Van Camp W. Yield enhancement genes: seeds for growth // Curr. Opin. Plant Biol. – 2005. – Vol. 16. – P. 147-153.
Meinke D.W., Cherry J.M., Dean C., Rounsley S.D., Koornneef M. Arabidopsis thaliana: a model plant for genome analy- sis. // Science. – 1998. – Vol. 282. – P. 679–682.
Zhang J.Z., Creelman R.A., Zhu J.-K. From laboratory to field. Using information from Arabidopsis to engineer salt, cold, and drought tolerance in crops // Plant Physiol. – 2004. – Vol. 135. – P. 615-621.
Turck F., Kozma S.C., Thomas G., Nagy F. A heat-sensitive Arabidopsis thaliana kinase substitutes for human p70s6k function in vivo // Mol. Cell. Biol. – 1998. – Vol. 18. – P. 2038-2044.
Ryabova L.A., Robaglia Ch., Meyer Ch. Target of Rapamycin kinase: central regulatory hub for plant growth and metabo- lism // J Exp. Bot. – 2019. – Vol.70, No.8. – P. 2211–2216.
Ren M., Qiu S., Venglat P., Xiang D., Feng L., Selvaraj G., Datla R. Target of rapamycin regulates development and ribo- somal RNA expression through kinase domain in Arabidopsis // Plant Physiol. – 2011. –Vol.155. – P. 1367-1382.
Roustan V., Jain A, Teige M., Ebersberger I, Weckwerth W. An evolutionary perspective of AMPK–TOR signaling in the three domains of life // J Exp. Bot. – 2016. – Vol. 67, No 13 – P. 3897-3907.
Nandagopal N., Roux P.P. Regulation of global and specific mRNA translation by the mTOR signaling pathway // Transla- tion. – 2015. – Vol. 3, No. 1.
Henriques R., Bögre L., Horváth B., Magyar Z. Balancing act: matching growth with environment by the TOR signalling pathway // J Exp. Bot. – 2014. – Vol.65, No.10 – P. 2691-2701
Caldana C., Martins M.C.M., Mubeen U., Urrea-Castellanos R. The magic ‘hammer’ of TOR: the multiple faces of a single pathway in the metabolic regulation of plant growth and development // J Exp. Bot. – 2019. – Vol.70. – P. 2217-2225.
Menand B., Desnos T., Nussaume L., Berger F., Bouchez D., Meyer C., Robaglia C. Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene // Proc. Natl. Acad. Sci. U.S.A. – 2002. – Vol. 99. – P. 6422–6427.
Rexin D., Meyer Ch., Robaglia Ch., Veit B. TOR signalling in plants // Biochem. J. – 2015. – Vol. 470. – P. 1–14.
Wolters H., Jürgens G. Survival of the flexible: hormonal growth control and adaptation in plant development // Nat. Rev. Genet. – 2009. – Vol. 10. – P. 305-317.
Creff A., Sormani R., Desnos T. The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent // Plant Mol. Biol. – 2010. – Vol. 73. – P. 533-546.
Ruvinsky, I., and Meyuhas, O. Ribosomal protein S6 phosphorylation: From protein synthesis to cell size // Trends Bio- chem. Sci. – 2006. – Vol. 31. – P. 342–348.
Meyuhas O. Ribosomal Protein S6 Phosphorylation: Four Decades of Research // Intl Review Cell & Mol. Biol. – 2016. – Vol. 320. – P. 1-33.
Turck F., Zilbermann F., Kozma S.C., Thomas G., Nagy F. Phytohormones Participate in an S6 Kinase Signal Transduction Pathway in Arabidopsis // Plant Physiol. – 2004. – Vol. 134. – P. 1527-1535.
Otterhag L., Gustavsson N., Alsterfjord M., Pical C., Lehrach H., Gobom J., Sommarin M. Arabidopsis PDK1: identifica- tion of sites important for activity and downstream phosphorylation of S6 kinase // Biochimie. – 2006. – Vol. 88. – P. 11–21.
Williams A.J., Werner-Fraczek J. Chang I.-F., Bailey-Serres J. Regulated Phosphorylation of 40S Ribosomal Protein S6 in Root Tips of Maize // Plant Physiol. – 2003. – Vol. 132. – P. 2086–2097.
Yamashita R., Suzuki Y., Takeuchi N., Wakaguri H., Ueda T., Sugano S., Nakai K. Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics // Nucl. Acids Res. – 2008. – Vol. 36, No. 11. – P. 3707– 3715.
Meyuhas O., Kahan T. The race to decipher the top secrets of TOP mRNAs // Biochim. Biophys. Acta. – 2015. – Vol. 1849.
– P. 801-811.
Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 // Nature. – 1970. – Vol. 227. – P. 680-685.
Sambrook J., Russel D.W. Molecular cloning: A laboratory manual: 3 volumes. – Third edition. – New-York: Cold Spring Harbor Laboratory Press, 2001.
Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding // Anal. Biochem. – 1976. – Vol.7, No.72. – P. 248-254.
