Structure‐function relationships of elongation factor Tu

The guanine‐nucleotide‐binding domain (G domain) of elongation factor 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 ( t 50% = 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 NH 4 + , 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 NH 4 + and Li + whilst Cs + is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3–5 mM, Mg 2+ being more effective than Mn 2+ and Ca 2+ . 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.