Reaction of ribosome-bound peptidyl transfer ribonucleic acid with aminoacyl transfer ribonucleic acid or puromycin.

Polylysyl transfer ribonucleic acid, free of lysyl-tRNA, has been isolated and its characteristics in an Escherichiu cofi polypeptide-synthesizing system in vitro have been studied. Ribosomes obtained from frozen E. coli cells were washed in 0.5 M NH&l and assayed at MgCla concentrations of at least 0.01 M. Binding of this peptidyl-tRNA to ribosomes occurs in the presence of templates containing 3 or more adenylic acid residues. p-Chloromercuriphenylsulfonate or lysyl-tRNA prevents this attachment. Binding occurs most effectively when both 30 S and 50 S ribosomal subparticles are present. Unlike lysyl-tRNA, bound polylysyl-tRNA reacts with puromycin in the absence of added GTP and supematant factors. The product of this reaction was shown to be polylysylpuromycin. Under s.nalogous conditions, a limited incorporation of lysine into bound polylysyl-tRNA can be shown. In this limited addition reaction, a polyadenylate template longer than three nucleotides is required. Chlortetracycllne is a strong inhibitor of the limited addition of lyslne to polylysyl-tRNA, but is without effect on the cleavage of polylysyl-tRNA by puromycln. Chloramphenicol, on the other hand, inhibits the reaction between polylysyl-tRNA and puromycin, but has little influence on the limited addition reaction. Gougerotin, a puromycin-like antibiotic, inhibits the puromycin reaction. Phenylalanyl-tRNA reacts poorly with puromycin in the presence of ribosomes and oligouridylate, whereas N-acetylphenylalanyl-tRNA is cleaved extensively, forming N-acetylphenylalanylpuromycin. Phenyllactyl-tRNA, although capable of binding to ribosomes, displays no detectable reactivity with puromycin. Chloramphenicol completely inhibits the cleavage of N-acetylphenylalanyltRNA by puromycin.