MUTANTS OF EF-TU DEFECTIVE IN BINDING AMINOACYL-TRANSFER-RNA

Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translati on in vitro. The substitutions are Leu(120)Gln, Gln(124)Arg and Tyr(16 0) (Asp or Asn or Cys). They mere selected by their kirromycin resista nt phenotypes and all substitutions are in domain I at the interface b etween domains I and LU of the EF-Tu GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k(cat)/K-M for the interaction between ternary complex and the programmed ribosome i s apparently reduced by the substitutions Leu(120)Gln, Gln(124)Arg and Tyr(160)Cys. Second, this reduction is caused by a defect in the inte raction between these EF-Tu variants and aminoacyl-tRNA during transla tion. Third, in four cases out of five the affinity of the complex bet ween EF-Tu . GTP and aminoacyl-tRNA is significantly decreased. The mo st drastic reduction is observed for the Gln(124)Arg change, where the association constant is 30-fold lower than in the mild-type case. Fou rth, missense errors are increased as web as decreased by the differen t amino acid substitutions. Finally, the dissociation rate constant (k d) for the release of GDP from EF-Tu is increased 6-fold by the Tyr(16 0)Cys substitution, but remains unchanged in the four other cases. The se results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.