Effects of Cations, Antibiotics and Other Agents on the Turnover of Guanosine‐Nucleotide · Elongation‐Factor‐G · Ribosome Complexes

The turnover of complexes containing elongation factor (EF) G, the ribosome and either GDP plus fusidic acid or guanyl‐5′‐yl methylene diphosphonate or guanyl‐5′‐yl imidodiphosphate has been measured by chasing the complexed, radioactive forms of these guanosine nucleotides with either an excess of unlabelled guanosine nucleotide or the antibiotic thiostrepton. With the three different complexes, the decay promoted by thiostrepton was substantially faster than that induced by the guanosine nucleotide and, under both decay conditions, no simple first‐order kinetics were observed. Assuming that the dissociation of the complexes proceeds via a guanosine‐nucleotide · EF‐G binary complex and a free ribosome, this result suggests that dissociation was reversible and that after it the inactivation of free ribosomes by thiostrepton was faster than the decay of the newly formed binary complex. Increasing the NH 4 + concentration from 10 to 180 mM or decreasing the Mg 2+ concentration from 30 to 10 mM during measurement of turnover accelerated the chase of [ 3 H]GDP from a [ 3 H]GDP · EF‐G · ribosome · fusidic‐acid complex. Since these variations did not affect the amount of complex present in solution, they evidently accelerated the reactions leading to formation of the complex. In contrast, increasing the NH 4 + concentration of the wash buffer used to isolate the complexes on nitrocellulose membranes sharply decreased their recovery. The antibiotics tetracycline, viomycin and several aminoglycosides that induce misreading (streptomycin, neomycin, kanamycin, tobramycin, gentamicin and paromomycin) inhibited the decay of the GDP · EF‐G · ribosome · fusidic acid complex, but other aminoglycosides like spectinomycin and kasugamycin had no effect. Several peptidyl transferase inhibitors (chloramphenicol, lincomycin, viridogrisein, griseoviridin, spiramycin, gougerotin and althiomycin), the EF‐Tu‐inactivating kirromycin and other antibiotics of less well defined mode of action (erythromycin, bottromycin and micrococcin) were also ineffective. Streptomycin and neomycin were effective at concentrations lower than 10 μM thus indicating that they were probably acting specifically and not as a result of a Mg 2+ ‐like action caused by their polycationic nature. The antibiotics did not completely block turnover and their effect could be explained by an increase in the affinity of the ribosome for the binary GDP · EF‐G complex. Since all the effective antibiotics bind to the 30‐S subunit and affect functions related to the ribosomal A‐site, the results emphasize the important role of this subunit for EF‐G interaction and suggest that EF‐G binds to the ribosome in a region near the 30‐S portion of the A‐site. A ternary complex containing Phe‐tRNA, EF‐Tu and GTP did not modify the chase rate of [ 14 C]GDP from a [ 14 C]GDP · EF‐G · ribosome · poly(uridylic acid) · fusidic‐acid complex.