Inhibitors of protein synthesis V Irreversible interaction of antibiotics with an initiation complex

Abstract

The initiation complex (t-complex) formed in a cell-free system (E. coli) from Ac-Phe-tRNA, poly(U) and washed ribosomes in the presence of initiation factors (ribosomal wash) and GTP, contains the Ac-Phe-tRNA bound quantitatively in a puromycin-reactive state. The t-complex is irreversibly inactivated by spiramycin with respect to its reactivity towards puromycin. The inactivated t-complex retains all of the Ac-Phe-tRNA bound, but it does not react with puromycin (2 × 10⁻³M) within 32 min at 25°. In the case of another inhibitor of protein synthesis, sparsomycin, the permanently “modified” t-complex not only retains all the bound Ac-Phe-tRNA but it can still react with puromycin. In the continuous presence of sparsomycin (1 × 10⁻⁷M) the bound Ac-Phe-tRNA reacts quantitatively at a rate which is one-tenth the rate at which the t-complex reacts with puromycin, at low (6.25 × 10⁻⁵ M) or high (2 × 10⁻³M) concentrations. These results are not in agreement with current views according to which sparsomycin binds to the ribosome reversibly at a single site with a K_I in the range of 10⁻⁶ 10⁻⁷M and according to which this site is at the A'-site (puromycin site) of peptidyi transferase.