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

Abstract

A simplified method for the separation of a kirromycin-sensitivetufB-encoded elongation factor Tu (EF-TuBs) from a kirromycin-resistanttufA product (EF-TuAr) was obtained by exploiting the specific increase of positive charges induced by the antibiotic, resulting in a retarded elution of kirromycin-bound EF-TuBs on ionic chromatography. The kirromycin-free EF-TuBs is active in poly(Phe) synthesis and shows similar properties to EF-TuAsBs. As expected for these two distinct species, the dissociation of the EF-TuArBs·GTP complex in the presence of kirromycin shows a biphasic curve; in contrast, a monophasic GTP dissociation rate was found for a combination of two mutated EF-Tu species. EF-TuArBo. revealing the existence of intermolecular interactions. These observations prove for the first time the existence of cooperative phenomena between EF-Tu species in vitro, as suggested earlier by in vivo experiments