Characterization of a kirromycin-resistant elongation factor Tu from Escherichia coli

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Abstract
The E. coli strain D2216 contains a kirromycin-resistant elongation factor Tu [EF-TuD2216]. This strain grows much more slowly than wild-type E. coli strains and contains < 1/2 the normal amount of EF-Tu. On isoelectric focusing, the whole cell lysate of strain D2216 and pure, crystalline EF-TuD2216 and pure, crystalline EF-TuD2216 comprises only a single species indistinguishable from wild-type EF-Tu. In poly(U) directed poly(Phe) synthesis, enzymatic binding of aminoacyl tRNA to the ribosome and susceptibility to trypsin digestion, EF-TuD2216 behaves similarly to the EF-Tu from wild-type strains. Kirromycin, which increases the sensitivity to trypsinization of wild-type EF-Tu, has no effect on mutant EF-Tu. In poly(U)-directed poly-(Phe) synthesis, partially trypsinized EF-TuD2216 displays a 7-fold reduction of its kirromycin resistance as compared to the intact EF-TuD2216. This is .apprx. 300 times less sensitive to the antibiotic than wild-type EF-Tu. The EF-TuD2216, purified and crystallized, exhibits a GTPase activity in the absence of any other physiological effector or kirromycin. This activity is not a contaminant, since it can be selectively stimulated by ribosomes and is inactivated by temperature exactly in the same way as the GDP binding activity of EF-TuD2216. As a consequence of the mutation, the catalytic center of EF-TuD2216-dependent GTP hydrolysis evidently undergoes spontaneous activation.