In Vivo Ribosome Binding Assay.

In vivo cross-linking of the translation factors to the ribosome was done as previously described (94). Briefly, cells grown to mid-log phase were chilled by addition of crushed ice (25% of total culture volume) and cross-linked by addition of formaldehyde to a final concentration of 1% relative to the original volume of the culture. After a 1-h incubation in an ice bath, cells were pelleted by centrifugation, washed with chilled water, and resuspended in the lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl, and 10 mM MgCl₂) supplemented with protease inhibitors and RNase inhibitor and frozen in liquid nitrogen. Cells were ruptured by grinding under cryogenic conditions. To monitor eIF1 binding to 40S, the lysate was cleared by centrifugation, and 20 U of optical density at 260 nm (OD₂₆₀) was loaded on a sucrose gradient (7.5 to 30% sucrose in the lysis buffer) and centrifuged in an SW41 rotor (Beckman Coulter) at 40,000 rpm for 5 h. Gradients were fractionated and analyzed by Western blotting using eIF1 antibody (a gift from A.G. Hinnebusch). To assess the binding of eEF2 to the ribosomes, 20 OD₂₆₀ of cleared lysate was loaded on 100 μL of sucrose cushion (1 M sucrose in lysis buffer) and centrifuged in TLA 100 (Beckman Coulter) for 2 h at 400,000 × g. The pellet was resuspended in lysis buffer plus SDS loading dye, and equal volumes of pellet and supernatant were analyzed by Western blotting using antibodies against eEF2 (Kerafast) and Rpl3 (Developmental Studies Hybridoma Bank).