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Mutants of EF-Tu defective in binding aminoacyl-tRNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
1996 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 382, no 3, 297-303 p.Article in journal (Refereed) Published
Abstract [en]

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 Leu120Gln, Gln124Arg 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 Leu120Gln, Gln124Arg and Tyr160Cys. 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 Gln124Arg change, where the association constant is 30-fold lower than in the mild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (k(d)) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160Cys 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.

Place, publisher, year, edition, pages
1996. Vol. 382, no 3, 297-303 p.
Keyword [en]
translation in vitro, kirromycin, EF-Tc, ternary complex
Identifiers
URN: urn:nbn:se:uu:diva-80296DOI: 10.1016/0014-5793(96)00184-6OAI: oai:DiVA.org:uu-80296DiVA: diva2:108210
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-14Bibliographically approved

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