THE STRUCTURAL AND FUNCTIONAL BASIS FOR THE KIRROMYCIN RESISTANCE OF MUTANT EF-TU SPECIES IN ESCHERICHIA-COLI

A structural and functional understanding of resistance to the antibio tic kirromycin in Escherichia coli has been sought in order to shed ne w light on the functioning of the bacterial elongation factor Tu (EF-T u), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage- mediated targeted mutagenesis, were studied, In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measur ed by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activi ties. The K'(d) values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in t he interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP . Evidence is presented that kirromycin binds to this interface of wil dtype EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu up on GTP hydrolysis. We conclude that the mutations result in two separa te mechanisms of resistance to kirromycin. The first inhibits access o f the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin a nd polypeptide chain elongation continues.