DISSOCIATION RATES OF PEPTIDYL-TRANSFER-RNA FROM THE P-SITE OF ESCHERICHIA-COLI RIBOSOMES

We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) alt ered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in t he wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild -type ribosomes is much stronger than to their A-site. Addition of str eptomycin dramatically reduces its affinity for the P-site, The S12 al terations make the P-site binding of peptidyl-tRNA much tighter, and t he S4, S5 alterations make it weaker than in the case of the wild-type . We find that when binding of peptidyl-tRNA to the A-site is weak, th en the affinity for the P-site is stronger, and vice versa, From these results, we formulate a hypothesis for the actions of streptomycin an d neomycin based on deformations of the 16S rRNA tertiary structure. T he results are also used to interpret some in vivo experiments on tran slational processivity.