(A) CBFs prevent 80S complex formation. [³²P]-labeled CAT mRNA (200,000 cpm) was incubated with cycloheximide (CHX) in the presence of FA or vehicle (MeOH) in rabbit reticulocyte lysates. Reactions were resolved by centrifugation through glycerol gradients. Following centrifugation, fractions were collected and the amount of radioactivity determined by liquid scintillation counting. The total counts recovered from each gradient and the percentages of mRNA bound in 80S complexes were: CAT mRNA/CHX + MeOH: 35,178 cpm, 19% binding; and CAT mRNA/CHX + FA: 37,655 cpm, 2% binding. (B) CBFs stimulate cross-linking of eIF4A to mRNA. Initiation factor preparations were cross-linked to [³²P]-cap-labeled mRNA in the absence (lane 2) or presence of ATP (lanes 1 and 3–6), 0.6 mM m⁷GDP (lane 3), 0.6 mM GDP (lane 4), MeOH (lane 5), or 50 μM FA (lane 6). Following nuclease digestion, samples were resolved by SDS-PAGE, and the gel was subjected to autoradiography. The position of migration of eIF4E, eIF4A, and eIF4B is indicated and is based on their known behavior in this assay (30). (C) CBFs stimulate the RNA-binding activity of eIF4AI_f. [³²P]-cap-labeled mRNA was cross-linked to recombinant eIF4AI_f in the presence of MeOH (lane 1), 50 μM FA (lane 3), or 50 μM silvestrol (lane 2). Following nuclease digestion, samples were resolved by SDS-PAGE, and the gel was subjected to autoradiography. (D) CBFs stimulate the RNA-binding activity of eIF4A_c. [³²P]-cap-labeled mRNA was cross-linked to eIF4F in the presence of MeOH (lane 1) or 50 μM FA (lane 2). Following nuclease digestion, samples were resolved by SDS-PAGE, and the gel was subjected to autoradiography. Cross-linking of a higher-molecular-mass species (>175 kDa) is indicated by “eIF4G ?” since it may reflect RNA-bound eIF4G.