Kirromycin&amp;#x2010;induced modifications facilitate the separation of EF&amp;#x2010;Tu species and reveal intermolecular interactions

Anborgh, Pieter H.; Swart, Guido W.M.; Parmeggiani, Andrea

doi:10.1016/0014-5793(91)80874-3

Cited by 22 publications

References 26 publications

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Pulvomycin-resistant mutants of E.coli elongation factor Tu.

et al. 1994

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This paper reports the generation of Escherichia coli mutants resistant to pulvomycin. Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring elongation factor (EF)‐Tu Arg230‐‐>Cys, Arg333‐‐>Cys or Thr334‐‐>Ala which confer pulvomycin resistance. These mutations are clustered in the three‐domain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF‐Tu. This result shares similarities with kirromycin resistance; kirromycin‐resistant mutations cluster in the domain 1‐3 interface. Since both interface regions are involved in the EF‐Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF‐Tu‐GTP to EF‐Tu‐GDP. The three‐domain junction changes dramatically in the switch to EF‐Tu.GDP; in EF‐Tu.GDP this region forms an open hole. Structural analysis of the mutation positions in EF‐Tu.GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues. All three mutations appear to destabilize the EF‐Tu.GTP conformation. Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance. This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin.

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Pulvomycin-resistant mutants of E.coli elongation factor Tu.

et al. 1994

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Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

et al. 1996

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This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF‐Tu‐GTP is strongly retarded, causing the Kd of EF‐ Tu‐GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP‐ and GDP‐bound EF‐Tu on native PAGE is increased. The stimulation of EF‐Tu‐GDP dissociation by EF‐Ts is inhibited. EF‐ Tu‐GTP can still form a stable complex with aminoacyl‐tRNA (aa‐tRNA), but it no longer protects aa‐tRNA against spontaneous deacylation, showing that the EF‐Tu‐GTP orientation with respect to the 3′ end of aa‐tRNA is modified. However, the EF‐Tu‐dependent binding of aa‐tRNA to the ribosomal A‐site is impaired only slightly by the antibiotic and the activity of the peptidyl‐transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C‐terminal incorporation of Phe into poly(Phe)‐tRNA bound to the P‐site is inhibited, an effect that is observed if Phe‐tRNA is bound to the A‐site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF‐Tu and the ribosomal A‐site directly, inducing an anomalous positioning of aa‐tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF‐Tu and the ribosome.

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Efficient Separation of Thermus aquaticus EF-Tu Functional Complexes

Степанов

Nyborg

2001

Biochemical and Biophysical Research Communications

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Kirromycin‐induced modifications facilitate the separation of EF‐Tu species and reveal intermolecular interactions

Cited by 22 publications

References 26 publications

Pulvomycin-resistant mutants of E.coli elongation factor Tu.

Pulvomycin-resistant mutants of E.coli elongation factor Tu.

Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

Efficient Separation of Thermus aquaticus EF-Tu Functional Complexes

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