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

Abstract

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.

Substitution of Arg230 and Arg233 in Escherichia coli Elongation Factor Tu Strongly Enhances Its Pulvomycin Resistance

“…GDP contributes to the inhibition of protein synthesis. In agreement with this is the recently presented study by Zeef et al (1994), in which it is shown that the pulvomycin-resistant mutant…”

“…Crystallographic studies (Hilgenfeld, R., unpublished results ; Zeef et al, 1994) have shown that Arg230 and Arg233 are part of the interface between the three domains.…”

Antibiotics and the Inhibition of Ribosome Function

“…Domain III of EF-Tu has been shown to be necessary for pulvomycin binding, leading to the speculation that the binding site of pulvomycin lies at the domain II-III interface [84]. A striking difference between pulvomycin and GE2770A relates to their resistance phenotype; pulvomycin sensitivity was found to be dominant to resistance [96], whereas in contrast GE2770A resistance was dominant over sensitivity [97,98]. This suggests that in the case of pulvomycin an additional mechanism must be operating other than simply limiting the availability of active ternary complex.…”

Inhibitory Mechanisms of Antibiotics Targeting Elongation Factor Tu

“…As a result, EF-Tu is prevented from delivering aa-tRNA to the ribosomal A site. Unexpectedly, however, pulvomycin sensitivity was found to be phenotypically dominant over resistance [25], suggesting that substrate limitation alone cannot be the exact mode of inhibition. Further research may be required to clarify these discrepancies.…”

“…stimulation of the replacement of GDP with GTP (Table 1), one can envisage the actions of pulvomycin as resembling those of the nucleotide-exchange factor, EF-Ts [24][25][26]. Like kirromycin, pulvomycin stimulates the GTPase activity of EF-Tu*GTP, although to a weaker extent [1].…”