FIG. 2.

Ribosomal-subunit anti-association activity of bacterially expressed recombinant yeast eIF6. eIF6 activity was measured by the ability of the protein to prevent the association of 40S and 60S ribosomal subunits at 5 mM Mg²⁺ to form 80S ribosomes, as described previously (37). Three reaction mixtures, each of 100 μl and containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 1 mM dithiothreitol, and 1.0 A₂₆₀ unit of Artemia salina 80S ribosomes, were prepared. Two of the reaction mixtures, A and B, contained no protein factors, while mixture C contained 1.25 μg of purified recombinant yeast eIF6 (Sephadex G-75 fraction). After incubation at 30°C for 5 min, the Mg²⁺ concentration of mixtures B and C was raised to 5 mM while that of mixture A was kept at 1 mM, and the incubation was continued for another 5 min at 37°C. Each reaction mixture was then chilled in an ice-water bath, loaded onto a 5-ml linear 5 to 30% (wt/vol) sucrose gradient containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM dithiothreitol, and 5 mM MgCl₂ (for mixtures B and C) or 1 mM MgCl₂ (for mixture A), and centrifuged for 90 min in an SW50.1 rotor at 48,000 rpm. Each gradient was fractionated and the A₂₅₄ profile was analyzed by using an UA-5 absorbance monitor. (A) No yeast eIF6 added, reaction at 1 mM Mg²⁺; (B) no yeast eIF6 added, reaction at 5 mM Mg²⁺; (C) yeast eIF6 added, reaction at 5 mM Mg²⁺.