Mutants of EF-Tu defective in binding aminoacyl-tRNA.
Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120 Gln, Gln124 Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III 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 is apparently reduced by the substitutions Leu120 Gln, Gln124 Arg and Tyr160 Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu.GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124 Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (kd) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160 Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.
Abdulkarim
F
F
Department of Molecular Biology, The Biomedical Center, Uppsala University, Sweden.
Ehrenberg
M
M
Hughes
D
D
D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010441:Peptide Chain Elongation, Translational / Q000502:physiology*; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics
D012346:RNA, Transfer, Amino Acyl; D011072:Poly U; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu