Abstract
Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.
Highlights
Elongation factors Tu and G (EF-Tu and elongation factor-G (EF-G))1 interact consecutively with the ribosomes during polypeptide synthesis
Choice of Mutations—The second G13V mutation in S. typhimurium EF-G markedly reduces fusidic acid (FA) resistance of the strain carrying the fus gene with the P413L mutation [13]
It was reported that the double A66V/T88A mutation confers FA resistance of E. coli EF-G [14]
Summary
Materials—All restriction endonucleases and T4 DNA ligase were from Promega. High fidelity Pfu Turbo DNA polymerase was purchased from Stratagene and used according to the manufacturer’s protocol. After the second round of PCR, the isolated DNA fragments were digested with NdeI and EcoRI enzymes and were cloned into the pET11c plasmid treated with corresponding endonucleases. By these means, the plasmids pETD109 and pETE119 were constructed. The GDP dissociation constant was determined in the reaction mixture (0.2 ml) in 50 mM Tris-HCl buffer, pH 7.6, 10 mM MgCl2, 1 mM dithiothreitol , and 70 mM NH4Cl containing 300 pmol of EF-G by titration with [3H]GDP (1.1 Ci/mmol) at 37 °C. The assay was carried out in the 100-l reaction mixtures containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 1 mM dithiothreitol , 70 mM NH4Cl, 0.4 mM GTP, 6 mM phosphoenol pyruvate, 1 g of pyruvate kinase, 20 pmol of E. coli. The inhibitory effect of fusidic acid on the turnover of GTP hydrolysis was followed by titration of the reaction mixture with FA (up to 5 mM) at the linear phase of the GTPase reaction
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