Inhibitory effect of diphtheria toxin on amino acid incorporation in Escherichia coli cell-free system.

The mechanism of action of diphtheria toxin in an Escherichia coli cell-free protein-synthesizing system was examined. When the washed ribosomes were incubated with toxin before addition of messenger ribonucleic acid (RNA), peptide syntheses of (14)C-phenylalanine directed by polyuridylic acid or phage R17 RNA were strongly inhibited by a small amount of toxin. Whereas, if the soluble fraction (105,000 x g supernatant fraction) was preincubated with toxin, no effect of toxin occurred either on the induced protein synthesis or on the activity of guanosine triphosphatase even in the presence of nicotinamide adenine dinucleotide. The binding of (3)H-formylmethionyl-transfer RNA to E. coli ribosomes directed by either R17 RNA or trinucleotide AUG was also decreased by toxin. These findings suggest that diphtheria toxin may prevent the binding of messenger RNA by successfully competing with the AUG for ribosomal binding sites. Sucrose-density gradient studies support this concept by showing the decrease in binding of (3)H-labeled R17 RNA to E. coli ribosomes exposed to toxin.