John R. Menninger

Ribosome editing and the error catastrophe hypothesis of cellular aging

Ribosome editing involves the dissociation during protein synthesis of inappropriate peptidyl-tRNAs, 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.

Tests of the ribosomal editing hypothesis: amino acid starvation differentially enhances the dissociation of peptidyl-tRNA from the ribosome.

Abstract Starving Escherichia coli for amino acids affected the dissociation of peptidyl-tRNAs from ribosomes. The frequency of dissociation of specific peptidyl-tRNA families responded differently to starvation for different amino acids rather than uniformly to the general condition of starvation. These results are interpreted in terms of the ribosomal editing hypothesis Menninger 1977. Starvation for some aminoacyl-tRNAs resulted in more opportunities for other aminoacyl-tRNAs to err, providing a greater amount of erroneous peptidyl-tRNA to be dissociated by the ribosomal editor. The details of the response of particular peptidyl-tRNA families to particular amino acid starvations show that a tRNA less able to decode correctly as an aminoacyl-tRNA is more likely to dissociate from the ribosome after peptide transfer. Many of the errors of translation thought previously to be rare may not have been detected in completed proteins because the ribosomal editor is most active against them. The results can also be interpreted as a specific regulatory response to amino acid starvation by a ribosome forced to pause during translation of non-essential proteins at codons whose aminoacyl-tRNAs are limiting, a model known as translational triage.

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

Inaccurate protein synthesis produces unstable β-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 β-galactosidase synthesized in the antibiotic-hypersensitive Escherichia coli strain DB-11 Met−. Newly synthesized β-galactosidase was more easily inactivated by high temperatures when synthesized by bacteria partially starved for arginine, threonine, or methionine. Simultaneous treatment with erythromycin or linocomycin yielded β-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, β-galactosidase was inactivated by high temperature more easily but this effect could not be reversed by erythromycin. The first arginine in β-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

SummaryPeptidyl-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 wildtype 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.

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

SummaryPeptidyl-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-resistent 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.