The puromycin reaction mediated by yeast ribosomes in high salt

Abstract

The extend of the reaction between puromycin and yeast peptidyl-tRNA prelabeled in vitro was determined by measuring the distribution of trichloroacetic acid precipitable material in isokinetic sucrose gradients in the presence of 0.5 M KCl.Thus it was found that increasing amounts of puromycin remove increasing amounts of peptidyl-tRNA from the 80S position in the gradient. The extend of the reaction, however, was independent of pretreatment of the ribosomes with inhibitors of the translocation indicating that peptidyl-tRNA at the donor and at the acceptor site of the ribosomes are equally accessible to puromycin at 0.5 M KCl.The exposure of both ribosomal binding sites to puromycin in high salt is accompanied by an enhanced reactivity of puromycin towards peptidyl-tRNA. The ED50 determined by measuring the inhibition by puromycin of the poly-U dependent phenylalanine incorporation drops from 5×10(-5) M below 250 mM KCl to 5×10(-6) M at 300 mM and higher concentrations of KCl.