Ribosome inactivation by ricin A chain: a sensitive method to assess the activity of wild-type and mutant polypeptides.
When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.
May
M J
MJ
Department of Biological Sciences, University of Warwick, Coventry, UK.
Hartley
M R
MR
Roberts
L M
LM
Krieg
P A
PA
Osborn
R W
RW
Lord
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003864:Depression, Chemical; D037121:Plant Lectins; D014176:Protein Biosynthesis / Q000187:drug effects; D012335:RNA, Ribosomal / Q000032:analysis*; D012339:RNA, Ribosomal, 28S / Q000032:analysis*; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D012156:Reticulocytes; D012270:Ribosomes / Q000187:drug effects*; D012276:Ricin / Q000235:genetics / Q000494:pharmacology*; D013045:Species Specificity; D014908:Triticum
D037121:Plant Lectins; D012335:RNA, Ribosomal; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D012276:Ricin