Viomycin does not stimulate the dissociation of peptidyl-tRNA.

Peptidyl-transfer RNA normally dissociates at a low rate from the ribosomes of Escherichia coli during protein synthesis but accumulates under nonpermissive conditions in cells with a temperature-sensitive allele (pthts) of the gene encoding peptidyl-transfer RNA hydrolase. The antibiotic-hypersensitive strain E. coli DB-11 with the pthts mutation was exposed to viomycin, then placed at nonpermissive temperatures. Under these conditions in the absence of drugs, peptidyl-tRNA accumulates, protein synthesis is inhibited and pthts cells die. When viomycin was present at sufficient concentration to arrest protein synthesis, cell death was not accelerated, error-inducing effects of streptomycin were not counteracted and, at high doses, cytoplasmic accumulation of peptidyl-transfer RNA was slowed down. Blocking the translocation of peptidyl-transfer RNA with viomycin did not stimulate its dissociation from ribosomes. Erythromycin-enhanced cell death was not affected by viomycin at doses sufficient to block amino acid incorporation, suggesting that short peptidyl-transfer RNAs could still be synthesized and dissociated from ribosomes.

Mechanism of inhibition of protein synthesis by macrolide and lincosamide antibiotics.

During protein synthesis on the ribosome, the growing peptide is linked covalently to a transfer RNA. With a certain probability this peptidyl-tRNA dissociates from the ribosome, whereupon it becomes susceptible to hydrolysis catalyzed by peptidyl-tRNA hydrolase. When placed at nonpermissive temperatures, mutant (pthts) Escherichia coli that are temperature-sensitive for the hydrolase will accumulate peptidyl-tRNA, suffer inhibition of protein synthesis, and eventually die. Treating cells with chloramphenicol before raising the temperature prevents cell death but erythromycin, other macrolides, and lincosamide antibiotics all enhance cell death. Accumulation of peptidyl-tRNA by pthts cells at high temperatures is blocked by chloramphenicol but enhanced by macrolides and lincosamides. The data are most consistent with macrolide and lincosamide antibiotics having as their primary mechanism of inhibition the stimulation of peptidyl-tRNA dissociation from the ribosome. Rather than blocking peptide bond formation or peptidyl-tRNA translocation from the A- to the P-site of the ribosome, these antibiotics allow the synthesis of small peptides which dissociate as peptidyl-tRNAs before being completed. Low doses of erythromycin and lincomycin stimulate preferentially the dissociation of peptidyl-tRNAs that are erroneous. Errors in proteins can be assessed by the time necessary to inactivate beta-galactosidase at > 55 degrees C. Whether erroneous peptidyl-tRNAs are induced by treating E. coli with streptomycin or ethanol, or by starving for an amino acid, the shortened time to inactivate beta-galactosidase is counteracted if the cells are simultaneously treated with erythromycin or lincomycin. In contrast, errors in beta-galactosidase caused by synthesis in the presence of canavanine, an arginine analogue, cannot be counteracted by the simultaneous presence of erythromycin. This result rules out any effect of the drug on post-translational mechanisms of error correction.

Erythromycin, lincosamides, peptidyl-tRNA dissociation, and ribosome editing.

Inaccurate protein synthesis produces unstable beta-galactosidase, whose activity is rapidly lost at high temperature. Erythromycin, lincomycin, clindamycin, and celesticetin were shown to counteract the error-inducing effects of streptomycin on beta-galactosidase synthesized in the antibiotic-hypersensitive Escherichia coli strain DB-11 Met-. Newly synthesized beta-galactosidase was more easily inactivated by high temperatures when synthesized by bacteria partially starved for arginine, threonine, or methionine. Simultaneous treatment with erythromycin or lincomycin yielded beta-galactosidase that was inactivated by high temperatures less easily than during starvation alone, an effect attributed to stimulation of ribosome editing. When synthesized in the presence of canavanine, beta-galactosidase was inactivated by high temperature more easily but this effect could not be reversed by erythromycin. The first arginine in beta-galactosidase occurs at residue 13, so the effect of erythromycin during arginine starvation is probably to stimulate dissociation of erroneous peptidyl-tRNAs of at least that length. Correction of errors induced by methionine starvation is probably due to stimulation of dissociation of erroneous peptidyl-tRNAs bearing peptides at least 92 residues in length. All the effects of erythromycin or the tested lincosamides on protein synthesis are probably the result of stimulating the dissociation from ribosomes of peptidyl-tRNAs that are erroneous or short.

Lincosamide antibiotics stimulate dissociation of peptidyl-tRNA from ribosomes.

At nonpermissive temperatures the peptidyl-tRNA hydrolase of pth(Ts) bacterial mutants is inactivated, and cells accumulate peptidyl-tRNA and die. Doses of erythromycin, lincomycin, or clindamycin that inhibited the growth of antibiotic-hypersensitive DB-11 pth+ cells accelerated the killing of DB-11 pth(Ts) cells at nonpermissive temperatures. Erythromycin and lincomycin also stimulated the accumulation of peptidyl-tRNA. Lincomycin and clindamycin stimulated peptidyl-tRNA dissociation from ribosomes.

Tests of the ribosome editor hypothesis. III. A mutant Escherichia coli with a defective ribosome editor.

Peptidyl-tRNA dissociates from the ribosomes of Escherichia coli during protein biosynthesis. The ribosome editor hypothesis states that incorrect peptidyl-tRNAs dissociate preferentially. Editing would therefore prevent the completion of proteins containing misincorporated amino acids. We have isolated a mutant strain of E. coli that dissociates some peptidyl-tRNAs at a fivefold lower rate than its parent strain, and that synthesizes significantly more erroneous complete proteins. This strain is also partially resistant to the antibiotic erythromycin, which in wild-type E. coli stimulates the dissociation of peptidyl-tRNA from ribosomes. The data suggest that in this mutant all peptidyl-tRNAs are bound to the ribosome more tightly than normally during protein synthesis. Because of the inverse correlation between the accuracy of synthesis of complete proteins and the rate of dissociation of peptidyl-tRNA from the ribosome, we propose that the mutant contains a defective ribosomal editor.

Functional consequences of binding macrolides to ribosomes.

Macrolide antibiotics bind to the large subunit of procaryotic ribosomes and perturb protein synthesis. There are two competing models to explain this perturbation: (1) shortly after initiation of the polypeptide chain, peptide bond formation and/or translocation is inhibited by the presence of macrolides that are bound in the ribosome 'tunnel' through which the nascent peptide travels; (2) bound macrolides loosen the interaction between the ribosome and peptidyl-tRNA, which therefore, dissociates with a higher probability. The former view cannot easily explain the observed enhancement by macrolides of the dissociation of peptidyl-tRNAs from ribosomes, while the latter view is consistent with the available data. Peptidyl-tRNAs are bound to ribosomes through non-specific and decoding-specific interactions. If macrolides preferentially weaken the non-specific interactions, a greater fraction of the binding energy will be due to decoding-specific interactions and better discrimination between erroneous and correct peptidyl-tRNAs should result. This idea has been tested with low doses of erythromycin, which was observed to counteract the error-inducing effects of streptomycin and of ethanol on the synthesis of beta-galactosidase by Escherichia coli. A specific error near the C-terminus of the enzyme was also responsive to this effect of erythromycin, which therefore must have exerted its influence long after the initiation of the polypeptide synthesis. These results are more easily explained by the idea that the primary mechanism of inhibition of protein synthesis by macrolides is to stimulate the dissociation of peptidyl-tRNA from the ribosome.

Dissociation of peptidyl-tRNA from ribosomes is perturbed by streptomycin and by strA mutations.

Peptidyl-tRNA may dissociate preferentially from ribosomes during protein synthesis when it is inappropriate to, does not correctly complement, the messenger RNA. To test this idea, growing cultures of Escherichia coli were treated with streptomycin to increase the frequency of errors during protein synthesis. Since the treated cells had a temperature-sensitive peptidyl-tRNA hydrolase and could not destroy dissociated peptidyl-tRNA, it was possible to measure the rate of its accumulation after raising the temperature to non-permissive conditions. Both low and high doses of streptomycin enhanced the rate of dissociation and accumulation of peptidyl-tRNA. The rank order of rates of dissociation/accumulation of various isoaccepting tRNA families was not significantly altered by the drug treatment. We concluded that streptomycin stimulated a normal pathway for dissociation of peptidyl-tRNA. Two streptomycin- resistant strains of E. coli had higher rates of dissociation of peptidyl-tRNA than did their sensitive parent strain. When treated with high doses of the drug, the resistant strains showed slightly reduced rates of dissociation of peptidyl-tRNA. These results were interpreted in terms of a two state, two site model for protein synthesis: streptomycin enhances the binding of aminoacyl-tRNA to a tight state of the ribosome A site; the strA mutation enhances translocation to a loose state of the ribosome P site.

Computer simulation of ribosome editing.

A stochastic model of protein synthesis was modified by including the process of dissociating peptidyl-tRNA from ribosomes. To simulate ribosome editing, the probability of dissociation was assumed to be high if the peptidyl-tRNA was erroneous; that is, if it resulted from transfer of a peptide to an aminoacyl-tRNA that was inappropriate relative to the mRNA codon. The effects of amino acid starvation on protein synthesis were simulated both by increasing the probability of such erring at and by reducing the conditional probability of elongation at "hungry" codons, those whose correct amino acid was in short supply. These probabilities were varied systematically to simulate tryptophan limitation during synthesis of coat protein from bacteriophage MS2. Significant reduction, during starvation, in the synthesis of complete coat protein required large reductions in the probability of elongation at hungry codons but only small increases in the probability of erring. Enhanced dissociation of peptidyl-tRNA during starvation, followed rapidly by dissociation of ribosomes from mRNA, led to reductions in mean polysome size, a result that had been interpreted by others as due to some effect of starvation on the initiation of protein synthesis. Results from experiments by Goldman (1982) on the cell-free synthesis of MS2 coat protein during tryptophan starvation could be mimicked in detail by the computer simulations. A simple competition between correct and erroneous amino acids was sufficient to explain the tryptophan dependence of complete coat protein and internal peptide syntheses. Values for the Michaelis constants were derived from the computer simulations.

Tests of the ribosome editor hypothesis. II. Relaxed (relA) and stringent (relA+) E. coli differ in rates of dissociation of peptidyl-tRNA from ribosomes.

Derivatives of isogenic stringent (relA+) and relaxed (relA) strains of Escherichia coli were compared in respect of rates of the dissociation of peptidyl-tRNA from ribosomes during protein synthesis. The derivatives both contained a mutant pth gene which rendered temperature-sensitive their peptidyl-tRNA hydrolase (E.C. 3.1.1.29) activities. After shifting from permissive 30 degrees C to non-permissive 40 degrees C, dissociated peptidyl-tRNA accumulated and was assayed chemically or by its cytotoxic effects. In unperturbed (except for the temperature shift) cultures the relA strain accumulated peptidyl-tRNA significantly more slowly than did its relA+ isogenic cousin. Both strains responded approximately equally to non-lethal doses of erythromycin or to starvation for amino acids. Both these perturbations enhanced the dissociation and accumulation of peptidyl-tRNA. While growing at 30 degrees C, both strains responded significantly to a nutritional downshift from growth in medium containing glucose plus amino acids to growth in medium containing only amino acids. Taken together the results suggested that different intracellular concentrations of ppGpp in unperturbed cells, attributable to the different relA alleles, could account for the differences in dissociation and accumulation of peptidyl-tRNA. Our observation of a lower rate of dissociation of peptidyl-tRNA in the relA strain, coupled with the reported lower intracellular ppGpp and lower accuracy of protein synthesis, is consistent with the idea that relA strains have less efficient ribosomal editing of erroneous peptidyl-tRNA.

Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

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 degrees 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 wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees 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.

Studies on termination of protein synthesis in wheat germ.

Oligophenylalanines are soluble in m-cresol, but oligophenylalanyl-tRNAs are not. This differential solubility can be used to assay oligophenylalanines released from tRNA during their synthesis by wheat germ extracts. When poly U is the message, virtually no free product appears. When poly A U (A < U) is used, a considerable amount of oligophenylalanines are released. The fraction of product released is approximately constant with time, implying that a steady-state is not achieved between initiation and release. The dependence of release on various reaction variables and the effects of several inhibitors on release indicate that the reaction is probably catalyzed by peptidyl transferase, in accord with the mechanisms described for mammals and prokaryotes.

Accumulation of peptidyl tRNA is lethal to Escherichia coli.

A mutant strain of Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase grows at 30 degrees C but, when shifted to 40 degrees C, 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.

Ribosome editing and the error catastrophe hypothesis of cellular aging.

Ribosome editing involves the dissociation during protein synthesis of inappropriate peptidyl-tRNA's, ones whose structure does not correctly complement the codon of the mRNA. This process is one of three stages in protein biosynthesis in which the frequency of errors in cellular proteins is controlled. These stages are reviewed and the implications of ribosome editing are described. A model for stability of the translation apparatus is criticized. Calculations using a revision of the model and experimentally reasonable values for the various parameters show varying time courses for error catastrophes.

Peptidyl transfer RNA dissociates during protein synthesis from ribosomes of Escherichia coli.

Growing cultures of mutant Escherichia coli with temperature-senstive peptidyl-tRNA hydrolase were shifted to nonpermissive 4o degrees. There followed a roughly linear increase in a fraction of isolated tRNA (over 50% after 20 min) whose amino acid-accepting activity was masked until treatment with active peptidyl-tRNA hydrolase. The ionophoretic mobility of amino acid label associated with this fraction could be altered by treatment with the hydrolase, trypsin, RNAse, and alkali. The rate of accumulation of this fraction could be altered by treating the growing cells with chloramphenicol, which reduced the rate, or erythromycin, which enhanced it. It is concluded that peptidyl-tRNA dissociates from ribosomes of the mutant cells during protein biosynthesis. The primary metabolic role of peptidyl-tRNA hydrolase is to prevent the accumulation of dissociated peptidyl-tRNA, which inhibits protein synthesis. The rate of dissociation of peptidyl-tRNA from ribosomes was estimated at between 1 per 90 and 1 per 2600 peptide elongation steps in the absence of antibiotics, depending on the level of inhibition of protein synthesis. After 20 min at 40 degrees, the size distribution of peptides found on tRNA was heterogeneous, with over 74% having a molecular weight greater than 8 X 10(2). The effect of erythromycin suggests that its mechanism of action is to destabilize the peptidyl-tRNA/ribosome interaction and thereby stimulate the dissociation of peptidyl-tRNA. The mechanism of inhibition of protein synthesis by accumulating peptidyl-tRNA and reasons why peptidyl-tRNA dissociates from ribosomes are discussed in terms of the current data.

Nepsilon-acetyllysine transfer ribonucleic acid: a biologically active analogue of aminoacyl transfer ribonucleic acids.

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.