Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes

Abstract

Light scattering was used to determine the effects of the bacterial initiation factors on the 70 S ribosome subunit equilibrium: 70 S ⇌ k 2 k 1 30 S + 50 s. The equilibrium proportion of associated ribosomes was studied as a function of IF-3, IF-1 and IF-2 § § Abbreviation used: IF, initiation factor; GMPPCP, 5′-quanylyl-methylene-diphosphonate. concentrations, and the rate constants κ 1 and κ 2 were determined in the presence of IF-3 and IF-1. Our results strongly support the view that IF-3-induced dissociation of the 70 S couple results from its binding to the free 30 S subunit, thus shifting the equilibrium toward dissociation. IF-3 causes κ 2 to decrease drastically, while κ 1 is unaffected; moreover, equilibrium values obtained with A-type ribosomes (tight couples) are consistent with binding of IF-3 to the 30 S subunit (association constant K 3 = 2·5 × 10 7 to 4·0 × 10 7 m −1) and with its exclusion from the 70 S ribosome. No significant variation of K 3 was found, either with temperature (25 to 37 °C) or with Mg 2+ concentration (2·2 to 5 m m). IF-1 was found to aid IF-3-induced dissociation by increasing the rate constants κ 1 and κ 2. IF-1 alone showed a limited dissociating activity, interpreted as due to its stronger binding to the 30 S than to the 70 S particle. Accordingly, κ 1 has a stronger dependence on IF-1 concentration than κ 2. The affinity of IF-3 for the 30 S subunit was observed not to vary significantly on addition of IF-1, when both factors were used in nearly stoichiometric amounts (not more than threefold excess over the ribosomes). When, on the contrary, IF-1 and IF-3 were present in large excess, dissociation was more efficient than expected from simple one to one binding of the factors to the ribosome. Streptomycin slowed the rate of dissociation by IF-3 and IF-1, but did not entirely suppress the effect of the factors. IF-2 was found to cause association of the ribosomal subunits; the effect is stronger and more specific in the presence of GMPPCP or GTP than in their absence. In the latter case, increase of the light-scattering signal probably involves some aggregation. In. the presence of GMPPCP, data are consistent with the following binding constants of IF-2: K 2 < 5 × 10 5 m −1 to the 30 S, and K 2′ = 1 × 10 7 to 1·5 × 10 7 m −1 to the 70 S ribosomes.