Partial release of AcPhe‐Phe‐tRNA from ribosomes during poly(U)‐dependent poly(Phe) synthesis and the effects of chloramphenicol

Poly(U)‐programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl‐tRNA analogue AcPhe‐tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe‐tRNA participate in poly(U)‐directed poly(Phe) synthesis (up to 65%) at 14 mM Mg 2+ and 160 mM NH + 4 . Here it is demonstrated that the apparently ‘inactive’ ribosomes (≧35%) are able to participate in peptide‐bond formation, but lose their nascent peptidyl‐tRNA at the stage of Ac(Phe) n ‐tRNA, with n ≥2. The relative loss of early peptidyl‐tRNAs is largely independent of the degree of initial saturation with AcPhe‐tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe) n ‐tRNA is reduced but still significant if more physiological conditions for Ac(Phe) n synthesis are applied (3 mM Mg 2+ , 150 mM NH + 4 , 2 mM spermidine, 0.05mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe‐tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe) 2 ‐tRNA nor peptide bond formation between AcPhe‐tRNA and Phe‐tRNA. The drug facilitates the release of Ac(Phe) 2–4 ‐tRNA from ribosomes at 14 mM Mg 2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).