SYNERGISM BETWEEN THE GTPASE ACTIVITIES OF EF-TU-CENTER-DOT-GTP AND EF-G-CENTER-DOT-GTP ON EMPTY RIBOSOMES - ELONGATION-FACTORS AS STIMULATORS OF THE RIBOSOMAL OSCILLATION BETWEEN 2 CONFORMATIONS

A remarkable positive cooperativity between the GTPase activities of E F-Tu and EF-G on empty ribosomes from Escherichia coli has been discov ered. This cooperativity implies a decrease of the corresponding appar ent K-M values of the empty ribosome fol either elongation factor: fro m more than 10 mu M to 0.5 mu M for EF-Tu.GTP by the addition of 0.25 mu M EF-G and from 0.7 mu M to 0.5 mu M for EF-G.GTP by the addition o f 3 mu M EF-Tu. In a further analysis Of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic ac id, tetracycline, pulvomycin and kirromycin appeared to inhibit the sy nergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-ment ioned antibiotics in a way surprisingly similar to that in the coupled system with mRSA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome a s revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-d enaturing conditions. Our experimental data and the kinetic analysis o f alternative models provide evidence that EF-Tu.GTP and EF-G.GTP inte ract sequentially with empty ribosomes that oscillate between two diff erent conformations, one for each elongation factor. Apparently, ribos omes have an intrinsic property for oscillation as normally observed d uring protein synthesis with a frequency paced by the events of tRNA b inding and translocation.