Inhibition of the peptide bond synthesizing cycle by chloramphenicol.
To study the mechanism by which chloramphenicol inhibits bacterial protein synthesis, we examined the kinetics of the puromycin-induced release of peptides from transfer ribonucleic acid (tRNA) in the presence and in the absence of chloramphenicol. Washed Escherichia coli ribosomes with nascent peptides which had been radioactively labeled in vivo were used for this study. When such ribosomes were incubated in the presence of 10 mug of puromycin per ml, approximately one-fourth of the radioactive peptide material was rapidly released from tRNA. This rapid, puromycin-dependent reaction is assumed to be equivalent to the peptidyl transferase reaction. Chloramphenicol inhibited the extent of the puromycin-induced release of peptides by only 50%, demonstrating that some of the peptide chains which are present on active ribosomes react with puromycin, even in the presence of chloramphenicol. The addition of the supernatant fraction and guanosine triphosphate (GTP) increased the extent of the puromycin-induced release; this additional release was completely inhibited by chloramphenicol. Peptidyl chains on washed ribosomes prepared from chloramphenicol-inhibited cells were not released by puromycin in the presence of chloramphenicol and reacted slowly with puromycin in the absence of chloramphenicol. The release of peptidyl groups from these ribosomes became largely insensitive to chloramphenicol after preincubation of the ribosomes with GTP and the supernatant fraction. We conclude that chloramphenicol does not inhibit the peptidyl transferase reaction as measured by the puromycin-induced release of peptides from tRNA, but rather inhibits some step in the peptide synthesis cycle prior to this reaction.
D001426:Bacterial Proteins / Q000096:biosynthesis*; D002701:Chloramphenicol / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D010452:Peptide Biosynthesis; D010455:Peptides / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D012329:RNA, Bacterial; D012343:RNA, Transfer; D012270:Ribosomes / Q000187:drug effects; D014316:Tritium
D001426:Bacterial Proteins; D010455:Peptides; D012329:RNA, Bacterial; D014316:Tritium; D011691:Puromycin; D002701:Chloramphenicol; D012343:RNA, Transfer; D007930:Leucine