Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate

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Abstract
Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met‐tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation‐factor‐2‐catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine‐containing peptides bound to 80S monosomes in ricin‐A‐chain‐inhibited and diphtheria‐toxin‐inhibited lysates, programmed with globin mRNA, revealed a predominance of Met‐Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin‐A‐chain‐inhibited and diphtheria‐toxin‐inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin‐A‐chain‐modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.