THE INFLUENCE OF DIFFERENT MODIFICATIONS OF ELONGATION FACTOR-TU FROM ESCHERICHIA-COLI ON TERNARY COMPLEX-FORMATION INVESTIGATED BY FLUORESCENCE SPECTROSCOPY

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu-GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu-GDP to 'active' EF-Tu-GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excission of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu GTP and E.coli Phe-tRNAPhe is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBo respectively, for [AEDANS-s2C]Tyr-tRNATyr are only slightly reduced and in the same range as trypsin-cleaved EF-Tu. © 1990 Oxford University Press.