When bottromycin A2 was added to an in vitro protein synthesis system carried out by naturally occurring polysomes, it inhibited protein synthesis effectively. Examination of the 3 steps of peptide chain elongation revealed that the binding of aminoacyl-tRNA to the polyribosomes was inhibited by bottromycin A2. In contrast, we concluded that the peptide bond formation and the translocation steps in this system were not inhibited by bottromycin A2 on the basis of the following observation: (1) the break-down of polysomes, which is dependent of EFG, puromycin and RR (ribosome releasing) factor, was insensitive to bottromycin A2; (2) the puromycin dependent release of polypeptide from polysomes, with or without EFG, was not inhibited by bottromycin A2. Thus bottromycin specifically interferes with proper functioning of the A sites of polysomes. This is consistent with results obtained using the model system with synthetic polynucleotides.
