Altered regulation of the guanosine 5'-triphosphatase activity in a kirromycin-resistant elongation factor Tu

Abstract

An Escherichia coli mutant elongation factor Tu (EF-TuD2216) which is resistant to the action of kirromycin displays a spontaneous GTPase activity in the absence of aminoacyl tRNA and ribosome-mRNA. This is the 1st example of an EF-Tu supporting GTPase activity in the absence of macromolecular effectors and/or kirromycin. This activity is elicited by increasing NH4+ concentrations. As additional effect, the mutation causes an increased affinity of EF-Tu for GTP. Ammonium dependence of the GTPase activity and increased affinity for GTP are 2 properties also found with wild-type EF-Tu in the presence of kirromycin. Thus, binding of kirromycin to wild-type EF-Tu and acquisition of kirromycin resistance introduce functionally related modifications. Kirromycin at high concentrations (0.1 mM) does not interact with mutant EF-TuD2216 .cntdot. GDP but still does with EF-TuD2216 .cntdot. GTP, in agreement with a previous finding that EF-Tu .cntdot. GTP is the preferential target of the antibiotic in the wild type. The GTPase activity of mutant EF-Tu in the presence of aminoacyl-tRNA and ribosome .cntdot. mRNA is much higher than with wild-type EF-Tu and also much less dependent on the presence of mRNA. Miscoding for leucine, measured as poly(U)-directed poly(Phe/Leu) synthesis at increasing Mg2+ concentrations, is identical for wild-type and mutant EF-Tu.