Structure-function relationships of elongation factor Tu.

Autor: Jensen, Michael, Cool, Robbert H., Mortensen, Kim K., Clark, Brian F.C., Parmeggiani, Andrea
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Zdroj: European Journal of Biochemistry; 6/15/89, Vol. 182 Issue 2, p247-255, 9p
Abstrakt: The guanine-nucleotide-binding domain (G domain) of elongation actor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufa gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 °C vs. 46°C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH4+, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH4+ and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu.
The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain · GTP complex and aa-tRNA. Our results indicate that the isolated G domain of EF-Tu is a suitable model for studying the basic properties of the guanine nucleotide interaction and the catalytic activity of a guanine-nucleotide-binding protein. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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