Mechanism of Action of Colicin E3. Effect on Ribosomal Elongation-Factor-Dependent Reactions

The effect of colicin E3 treatment on the activity of Escherichia coli ribosomes in the partial reactions of the elongation cycle was studied. The reactions associated with elongation factor T (EF-T = EF-Tu · EF-Ts) were found to be affected by treatment with colicin E3: at 5 mM Mg2+ the EF-T-dependent GTPase reaction was stimulated 6–7-fold and enzymatic binding of Phe-tRNA was reduced 4-fold, while at 25 mM Mg2+ neither the GTPase reaction nor binding of Phe-tRNA was affected. Since the EF-T-dependent GTPase reaction became progressively less dependent on poly(U) with increasing [Mg2+] while dependence on Phe-tRNA persisted throughout, this effect could be mediated by a distorted configuration of the mRNA binding site at low [Mg2+] following cleavage near the 3′-OH terminus of 16-S ribosomal RNA. This possibility is also supported by the finding that poly(phenylalanine) synthesis was strongly inhibited at low (5–8 mM) [Mg2+], i.e. in the range optimal for this reaction, while at > 15 mM Mg2+ inhibition disappeared, presumably because at high [Mg2+] the ribosomal mRNA binding site does not have the same requirements for the intact 3′-OH moiety of the 16-S ribosomal RNA as at low Mg2+ concentration. Ribosomal EF-G GTPase activity was in no way changed by the colicin treatment even when mRNA and tRNA stimulated (low [Mg2+]) or inhibited (high [Mg2+]) this reaction. Chromatography of ribosomes on a Sepharose 4B column equilibrated with [14C]colicin E3 showed physical interaction between [14C]colicin E3 and ribosomes. Aminoacyl-tRNA bound to the ribosomal A or P-site protected the ribosome against inactivation by the colicin. The state of association of the ribosomal subunits was not affected if colicin E3 was administered in vitro, while treatment in vivo resulted in ribosomes with decreased ability to associate but with essentially unaltered functional properties.