In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40°C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wildtype cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30°C). Since peptidyl-tRNA hydrolase in the mutant cells is inactivated rapidly and irreversibly at 40°C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation.The mechanism of action of the macrolide antibiotics has been a matter of controversy for some time (7,31). A few years ago, we reported data concerning a novel effect of erythromycin on protein synthesis in vivo, namely, the enhancement of the dissociation of peptidyl-tRNA from ribosomes (J. R. Menninger, Fed. Proc. 33:1335. This phenomenon was subsequently verified in vitro (24). Our in vivo studies have now been extended to two other antibiotics of the macrolide group, carbomycin and spiramycin.We used a strain of Escherichia coli (ts8) with a mutation in the structural gene (pth) for peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase, EC 3.1.1.29) which renders that enzyme activity temperature sensitive. Peptidyl-tRNA hydrolase normally acts in the cell to catalyze the hydrolysis of intact peptides from peptidyltRNA that has dissociated from ribosomes during protein synthesis. A temperature-sensitive hydrolase activity allows measurement of the dissociation of peptidyl-tRNA from ribosomes at 40°C since the first step in the scavenging pathway for dissociated peptidyl-tRNA is blocked by the high temperature (18). Shortly after cultures growing at 30°C are shifted to the nonpermissive temperature (40°C), protein synthesis is inhibited (1, 18), and the cells die; i.e., they are unable to form a colony at permissive temperatures (21). All of these effects were assayed in the presence of erythromycin, carbomycin, and spiramycin at doses chosen, after the evaluation of several concentrations, so that no lethal effects of the drugs were observed at permissive temperatures.The results presented below show that all three of these antibiotics stimulated the dissociation of peptidyl-tRNA from ribosomes of E. coli when given at doses that inhibited the gr...

No abstract

Unfractionated Escherichia coli tRNA has been aminoacylated with lysine and preferentially acetylated at the epsilon-amino nitrogen of lysine by reaction with N-acetoxysuccinimide. After treatment with peptidyl-tRNA hydrolase, 90% of the aminoacylated tRNA molecules were Nepsilon-acetyl-Lys-tRNA. Post-ribosomal supernatant enzymes would not deacylate Nepsilon-acetyl-Lys-tRNA in the presence of AMP and PPi, even though such mixed enzymes could acylate, with lysine, tRNA which had been exposed to the acetylation reaction conditions. Poly(rA) stimulated the binding of Nepsilon-acetyl-Lys-tRNA to E. coli ribosomes. At the ribosome and tRNA concentrations used, Nepsilon-acetyl-Lys-tRNA was bound nearly as well as Lys-tRNA at 30 mM Mg2+; at 10 mM Mg2+, the analogue was bound one-half as well as Lys-tRNA. Both Lys-tRNA and Nepsilon-acetyl-Lys-tRNA reacted only slightly with puromycin at either 10 or 30 mM Mg2+. When Lys-tRNAE. coli or Nepsilon-acetyl-Lys-tRNAE. coli were added to rabbit reticulocyte cell-free protein synthesizing incubations, the incorporation of either amino acid into protein was complete within 5 min. The final incorporation level of the analogue was 82% that of the unmodified lysine. After protein synthesized in the presence of Nepsilon-acetyl-[14C]Lys-tRNA had been digested enzymatically to single amino acids, ion-exchange chromatography and paper electrophoresis showed that nearly all of the radioactivity was present as Nepsilon-acetyllysine. Gel filtration of the post-ribosomal supernatant revealed that most of the Nepsilon-acetyllysine radioactivity cochromatographed with tetrameric hemoglobin.

A mutant strain of Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase grows at 300C but, when shfted to 400C, dies at rates affected by physiological, pharmacological, and genetical perturbations. The rate of killing correlates with the relative accumulation of peptidyl-tRNA, suggesting that it is responsible for the death of the cells.