Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain.

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 (t50% = 41.3 degrees C vs. 46 degrees 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, 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the ATPase and GTPase activities of elongation factor 3 (EF-3) purified from yeasts.

Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe. Both of the proteins consist of a single peptide chain. The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes. The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP. Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2. Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2. Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.

Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins.

Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP-binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product.