INHIBITION BY PACTAMYCIN OF INITIATION OF PROTEIN SYNTHESIS . BINDING OF N-ACETYLPHENYLALANYL TRANSFER RIBONUCLEIC ACID AND POLYURIDYLIC ACID TO RIBOSOMES

The inhibition by pactamycin of polyphenylalanine synthesis by extracts from Escherichia coli is greatest at low Mg2+ concentrations where chain initiation by either N-acetylphenylalanyl transfer ribonucleic acid or phenylalanyl transfer ribonucleic acid is rate limiting. This inhibition is decreased by prior incubation of the S-30 extract at 4.5 mM Mg2+ with N-acetylphenylalanyl transfer ribonucleic acid but not with phenylalanyl transfer ribonucleic acid. Preincubation of the S-30 with pactamycin results in increased inhibition of polypeptide formation, while the delayed addition of pactamycin until after initiation is finished has no effect on polypeptide elongation. At low Mg2+ concentrations the binding of N-acetylphenylalanyl transfer ribonucleic acid to ribosomes, which depends upon initiation factors and guanosine triphosphate, is significantly inhibited by the antibiotic (10-6 M). Pactamycin induces the release of prebound N-acetylphenylalanyl transfer ribonucleic acid but does not directly inhibit deacylation caused by puromycin. The effect of pactamycin on the binding reaction is inversely related to the Mg2+ and directly related to the NH4+ concentrations. Streptomycin at low levels and chlortetracycline at high levels also interfere with the binding reaction. Zonal centrifugation analyses of binding incubations reveal that the bound radioactive N-acetylphenylalanyl transfer ribonucleic acid sediments more rapidly during the course of the reaction. In the presence of pactamycin, radioactivity sediments more slowly. While such “particles” may be 50S-30S complexes with varying compactness of structure, it is possible that they represent 70S ribosomes which are disassociating during sedimentation. Pactamycin is viewed as interfering with compact 70S ribosome formation, resulting in more ready disassociation into the component subunits. This effect is accentuated by low Mg2+ and high NH4+ concentrations which also decrease the stability of the binding and weaken the attraction between the ribosomal subunits. At 20 mM Mg2+ or 10 mM spermidine the pactamycin effect is lost. Binding of polyuridylic acid to ribosomes in the presence, but not in the absence, of initiation factors depends upon the addition of N-acetylphenylalanyl transfer ribonucleic acid and guanosine triphosphate. Only the former type of binding of polyuridylic acid is inhibited by pactamycin. It is concluded that pactamycin alters the structure of the initiation complex so as to lead to its destabilization and dissociation. © 1969, American Chemical Society. All rights reserved.