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It has been formerly shown that low concentrations of Mg 2+ , as are used to dissociate ribosomes into subunits, render the ribosomes inactive in binding of aminoacyl-transfer RNA. Activity is restored on heating the ribosomes under appropriate salt conditions. We have now studied the binding of aminoacyl-tRNA to fully activated ribosomes thus eliminating any possible activation during the binding reaction. The requirements and kinetics of the reaction as revealed are therefore those of the binding reaction and not of the activation step. The major findings were as follows. 1. (i) Non-enzymic binding of Phe-tRNA to 30 s subunits proceeds efficiently in the cold and at Mg 2+ concentrations as low as 5 mM. 2. (ii) The instability of the 30 s messenger RNA-aminoacyl-tRNA complex is suggested to be due to transformation of the 30 s subunits into a form incapable of further binding. This inactivation occurs during the binding reaction and depends on it. 50 s subunits prevent the inactivation and restore binding ability to seemingly inactive 30 s subunits. The observed stimulation of aminoacyl-tRNA binding to 30 s subunits by 50 s subunits may thus be due to stabilization of the active form of the ribosome by 50 s subunits and not necessarily to the formation of an additional binding site. 3. (iii) 50 s subunits inhibit the initial rate of binding of different aminoacyl-tRNA's, indicating that such inhibition is not a unique property of fMet-tRNA binding. 4. (iv) T factor stimulation of aminoacyl-tRNA binding is related to the inhibition of the reaction by 50 S subunits. The results indicate that binding to isolated 30 s subunits represents under the given conditions the full binding potential of the ribosomes. 50 s subunits suppress this binding and the presence of T factor restores it to the level seen with 30 s subunits alone. Thus, the Tu-GTP-aminoacyl-tRNA complex appears to overcome the inhibition exerted by 50 s subunits.

β-Chelocardin binds to both 30-S and 50-S ribosomal subunits. The amount of this antibiotic bound by the 50-S subunits is half that bound by the 30-S subunits. Chlorotetracycline competes with this antibiotic for the binding sites on both ribosomal subunits. β-Chelocardin interferes with the binding of amino acyl-tRNA to the 30-S subunits but not to the 50-S subunits. These data indicate that the mode of action of β-chelocardin is identical to that of tetracycline.