Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits.

The magnesium dependence of the Escherichia coli ribosomal subunits association has been investigated by the stopped-flow technique using isolated 30S and 50S particles depleted of polyamines and any initiation factor. Binding of the fluorescent probe bis(8-anilino-1-naphthalenesulfonate) to the ribosomal proteins occurs through biphasic kinetics. A dark reaction corresponding to a very rapid, reversible complexation of the dye molecule is followed by a slow photochemical reaction that gives rise to irreversible addition of the probe. Only the 30S subparticle exhibits a magnesium-dependent conformational change from the kinetic analysis of the dark reaction. The 70S formation kinetics are limited by a conformational change of the 30S subunit if this particle is depleted of Mg2+ (1 mM Mg2+/50 mM K+), while its activated structure is restored by incubation with 8 mM Mg2+/50 mM K+. No rate-limiting conformation rearrangement of the 50S subunit could ever be evidenced. The Mg2+ dependence of the association kinetics of preactivated ribosomal particles is satisfactorily explained by electrostatic effects and/or formation of salt bridges, in agreement with the results of Wishnia and co-workers (Wishnia, A. Boussert, A., Graffe, M., Dessen, P., and Grunberg-Manago, M. (1975), J. Mol. Biol. 93, 499). Equilibrium studies indicate that the ribosomal preparations we used are of B type, according to Debey et al. (Debey, P., Hui Bon Hoa, G., Douzou, P., Godefroy-Colburn, T., Graffe, M., AND Grunberg-Manago, M. (1975) Biochemistry 14, 1553). The addition of spermidine results in a drastic fall of the need of Mg2+ for association, but it does not allow conversion of B-type particles into A-type ones at 25 degrees C. In addition to that, some 30S-bound spermidine appears to be involved directly in the coupling reaction.