SUBSTITUTION OF ARG230 AND ARG233 IN ESCHERICHIA-COLI ELONGATION-FACTOR TU STRONGLY ENHANCES ITS PULVOMYCIN RESISTANCE

Pulvomycin is a strong inhibitor of protein synthesis, known to preven t the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu GTP ). Recently, three pulvomycin-resistant mutant strains have been isola ted by targeted mutagenesis of the tufA gene resulting in EF-Tu substi tutions at positions 230, 333 or 334. In order to analyze the function s of arginine residues located in domain II, with respect to pulvomyci n resistance and the interaction with aminoacyl-tRNA, we have investig ated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two m utants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affini ty chromatography. In this study, we describe the in vitro characteriz ation of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increa ses the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rath er than by EF-Tu GTP pulvomycin. Moreover, the mutant [R230V, R233F]EF -TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.