The association-dissociation kinetics of ribosomes from Escherichia coli have been studied under various conditions in a light-scattering stopped-flow apparatus. The dissociation reaction at 2 mg/ml at 25 °C, induced by lowering the MgCl2 concentration from 18 to 3 mM, can best be described by three independent first-order processes with rate constants of 15 s-1, 0.9 s-1, and 3 × 10-2 s-1 the slowest process comprising about 60% of the overall reaction. The fraction of ribosomes dissociating with the fastest rate (15 s-1) is concentration dependent and becomes negligible at 0.1 mg/ml. Ribosomes treated with puromycin also show three dissociation rates with essentially the same rate constants as the nontreated samples. The dissociation induced by a high KCl concentration (0.85 M KC1, 18 mM MgCl) also shows three first-order phases with the same rate constants as for the dissociation induced by lowering the MgCl2 concentration. The formation of 70S ribosomes from 30S and 50S subunits, induced by increasing the MgCl2 concentration from 2 to 21 mM, follows second-order biphasic kinetics. A detailed analysis of the kinetic results shows that the two principal ribosomal forms must have one type of subunit in common. When the association data are analyzed assuming that the kinetic heterogeneity arises from two forms of only one subunit, the rate constants are found to be 6.4 × 106 and 1.05 × 106 M-1 s-1. Sequential flow experiments show that the rapid and slow association species are to be identified, respectively, with phases II (0.9 s-1) and III (0.03 s-1) of dissociation. Relaxation measurements show that these correspond to type B (“loose”) and A (“tight”) ribosomes, respectively. Tight and loose ribosomes were isolated by sucrose density centrifugation, and dissociation and association kinetic studies confirmed the above assignments. Furthermore, the rate constants for these ribosomes agreed within experimental error with the rate constants derived from analysis of the multiphasic kinetic data. The association rate constants are for ribosomes dissociated by dilution with the appropriate buffer immediately before recording the kinetics of association. Ribosomes dissociated by dialysis overnight against 2 mM MgCl2 show an association rate constant for the slower association reaction (type-A ribosome) that is about four times smaller, whereas the rate constant for the faster process is roughly the same. The activation energies of the dissociation reactions, whether induced by lowering the MgCl2 concentration or increasing the KC1 concentration, and the association reaction induced by increasing the MgCl2 concentration are less than 3.5 kcal/mol. The rate constants of the dissociation at 3 mM MgCl2 and of the association reaction at 21 mM MgCl2 do not vary between pH 7.2 and 8.4. When 30S and 50S subunits are flowed against buffer containing 20 mM spermidine, the association process is monophasic, with an association constant k = 6 × 106 M-1 s-1. Approach-to-equilibrium studies show that kdissoc varies with (Mg2+)n and kdissoc varies with (Mg2+)n (n = 2.3-3.7, m = 2-3) for 4 mM < (Mg2+) < 8 mM.
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