THE G222D MUTATION IN ELONGATION-FACTOR TU INHIBITS THE CODON-INDUCEDCONFORMATIONAL-CHANGES LEADING TO GTPASE ACTIVATION ON THE RIBOSOME

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutati on G222D is unable to hydrolyze GTP on the ribosome and to sustain pol ypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not c hanged significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-st eady-state kinetic study of the binding of the ternary complexes of mi ld-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of p oly(U)-programed ribosomes, The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wildtype EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affe cted by the mutation, Codon recognition following initial binding is a lso not affected by the mutation, The main effect of the G222D mutatio n is the inhibition, at low Mg2+ concentration, of codon-induced struc tural transitions of the tRNA and, in particular, their transmission t o EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-sit e binding, Increasing the Mg2+ concentration to 10 mill restores the c omplete reaction sequence of A-site binding at close to wild-type rate s. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with n egative charges either of the 3' terminus of the tRNA, bound in the vi cinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribos ome, Increasing the Mg2+ concentration appears to overcome the inhibit ion by screening the negative charges.