Figure 8.

Differences between 48S complexes assembled on cap-proximal initiation codons and “complex I” aberrantly assembled on natural capped globin mRNA in subunit joining activities and in protection by 60S subunits from eIF1-induced destabilization. (A,D) Sucrose density gradient centrifugation of 48S and 80S complexes assembled on [³²P]-labeled (A) GG-AUG-(CAA)n-GUS mRNA and (D) globin mRNA from purified translation components as indicated. Sedimentation was from right to left. (B,E) Effect of inclusion of eIF1 at indicated times to assembly reactions that contained (B) G-AUG-(CAA)n-GUS mRNA and (E) β-globin mRNA, GTP, aminoacylated tRNA, and other translation components as indicated. (C) Effect of inclusion of increasing amounts (0.05 μg, lanes 3,8; 0.15 μg, lanes 4,9; 0.45 μg, lanes 5,10; 1 μg, lanes 6,11) of eIF1 in assembly reactions that contained G-AUG-(CAA)n-GUS mRNA, GTP, aminoacylated tRNA, and other translation components as indicated. Lanes 2 and 7 did not contain added eIF1.