Effect of Puromycin Analogues and Other Agents on Peptidyl-Puromycin Synthesis on Polyribosomes

The incorporation of [3 H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide bond synthesis. Several analogues of puromycin were found to react with polyribosomes from both bacteria and rat liver. TheKm for puromycin is 4 μM with bacterial polyribosomes; under the same conditions, theKi for ψ-hydroxy-puromycin (6-dimethylamino-9-[3-(l -β-phenyllactylamino)-3-deoxy-β-d -ribofuranosyl] purine) is 240 μM and for a carbocyclic analogue of puromycin (6-dimethylamino-9- {R- [2R-hydroxy-3R- (p-methoxyphenyl-l -alanylamino)]-cyclopentyl}purine) is 1 μM. Both were found to be competitive inhibitors of puromycin. TheKm for C-A-C-C-A(Phe) is 250 μM. In addition, the dissociation constant for C-A-C-C-A(Phe) binding to washed ribosomes was found to be 1 and 0.03 μM in the absence and presence, respectively, of 20% (vol/vol) ethanol. The results with these analogues lead to the following conclusions. Substitution of a hydroxyl group for the α-amino group of puromycin results in an active analogue with about one-sixtieth the affinity of puromycin in the reaction. Omission of the 5′-hydroxymethyl group or substitution of the furanosyl ring oxygen by a carbon atom in the carbocyclic analogue reduces its activity compared with puromycin only slightly. Additionally, the relatively highKm for C-A-C-C-A(Phe) as an acceptor compared with puromycin suggests the existence of a protective mechanism on polyribosomes, which prevents aminoacyl-transfer ribonucleic acid (tRNA) free in solution from stripping nascent chains from polyribosomes so that only aminoacyl-tRNA bound to ribosomes through the appropriate coding mechanism can form a peptide bond.