Reaction of puromycin with N-acetylphenylalanyl-tRNA on ribosomes reassociated from escherichia coli ribosomal subunits

Abstract

N-Acetylphenylalanylpuromycin synthesis catalyzed by ribosomes reassociated from 50-S and 30-S subunits was studied. N-Acetylphenylalanylpuromycin was formed even at o° by addition of puromycin to a mixture of 50-S ribosomal subunits and a preparation of 30-S ribosomal subunits, poly(U) and N-acetylphenylalanyl-tRNA previously mixed at o° after preincubation under suitable conditions. To obtain maximal formation of N-acetylphenylalanylpuromycin by reassociated ribosomes, the 50-S ribosomal subunits had to be preincubated at a sufficient concentration of NH 4 + (or K +) at a suitable temperature (about 37°), but a change in the concentration of Mg 2+ showed no significant effect. The preincubation of 30-S ribosomal subunits did not require a particular concentration of NH 4 + (or K +) but a sufficient Mg 2+ concentration was necessary. Using this technique we were able to study the effect of environmental conditions on the activity of respective ribosomal subunits in peptide synthesis. The 50-S and 30-S subunits preparations were obtained from ribosomes of parent Escherichia coli Q13 strain and from its mutant ribosomes which have an altered specific protein component in the 50-S subunit and a high monovalent cation (K + or NH 4 +) dependency in the peptidyl transfer reaction. The hybrid ribosomes consisted of Q13 30-S subunits and the mutant 50-S subunits showed distinctly higher monovalent cation dependency than the hybrid ribosomes from Q13 50-S subunits and the 30-S subunits of the mutant ribosomes, suggesting that alteration of 50-S ribosomal subunits induces alteration of their peptidyl transferase activity.