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

Abstract

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 Mg2+ 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 Mg2+, 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 Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys). Copyright © 1990, Wiley Blackwell. All rights reserved