Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation.
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.
Osborn
R W
RW
Department of Biological Sciences, University of Warwick, Coventry, England.
Hartley
M R
MR
D000818:Animals; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D004167:Diphtheria Toxin / Q000494:pharmacology; D005914:Globins / Q000096:biosynthesis*; D066298:In Vitro Techniques; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects*; D010444:Peptide Elongation Factor Tu / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D011687:Purines / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D012339:RNA, Ribosomal, 28S / Q000187:drug effects; D012358:RNA, Transfer, Met / Q000187:drug effects; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects*; D012276:Ricin / Q000494:pharmacology*; D014178:Translocation, Genetic / Q000187:drug effects*
D004167:Diphtheria Toxin; D011687:Purines; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D012358:RNA, Transfer, Met; D005914:Globins; D012276:Ricin; D010444:Peptide Elongation Factor Tu