Molecular Properties of Two Mutant Species of the Elongation Factor Tu

Abstract

The molecular properties of two mutant species of the elongation factor Tu (EF‐Tu), derived from either tuf A or tufB, have been studied. One, designated EF‐TuA_R, is the product of a kirromycin‐resistant tufA gene. The other designated EF‐TuB_o is a tufB product and is present in a kirromycin‐resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF‐TuA_R species. EF‐TuA_R has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tufB gene has been inactivated by an insertion of the bacteriophage Mu. EF‐TuB_o has been isolated from LBE 2012 together with EF‐TuA_R in a 1:1 mixture. Fractionation of this mixture on DEAF‐Sephadex A‐50 resulted in an enrichment of EF‐TuB_o of about 80%. The properties of EF‐TuA_R and EF‐TuB_o have been compared to those of a kirromycin‐sensitive species designated EF‐TuA_S, which was isolated from LBE2045 by transduction of wild‐type tufA. We show here that all three EF‐Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF‐Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF‐TuA_S (wild‐type tufA gene product) to be retained on, and thus block, the ribosome. (b) EF‐TuA_R fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EFTuB_o fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF‐TuA_R (LBE2012) is kirromycin resistant.