Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits

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Abstract
The Mg2+ dependence of the E. coli ribosomal subunits association was investigated by the stopped-flow technique using isolated 30S and 50S particles depleted of polyamines and initiation factors. 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 Mg2+-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 was ever evidenced. The Mg2+ dependence of the association kinetics of preactivated ribosomal particles is satisfactorily explained by electrostatic effects and/or formation of salt bridges. Equilibrium studies indicate that the ribosomal preparations are of B type. 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.degree. C. Some 30S-bound spermidine may be involved directly in the coupling reaction.