All steps were performed in a buffer of 50 mM Hepes pH 7.3, 50 mM KCl, 10 mM Mg(C2H3O2)2, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamide. Of note, 40 U RNase inhibitor (NEB) were added per 1 mg/mL of total protein concentration. Cell extracts from S. cerevisiae were precleared with 60 µL of paramagnetic streptavidin-conjugated magnetic beads (Dynabeads M-280; Invitrogen) for 1 h at 4°C to decrease the amount of protein bound nonspecifically to the beads. S1-tagged 5′UTRPab1 RNAs alone and the 5′UTRPab1-43S PIC were incubated with the beads for 2 h at 4°C in the buffer consisting of 50 mM Hepes, pH 7.3, 100 mM KCl, 2 mM MgCl2, and 2 mM DTT. The formation of the 43S PIC was carried out as described previously (Acker et al. 2007). To facilitate ribosome-loading, 5′UTRPab1-construct1 was used in which the RNA sequence downstream from the start codon AUG was replaced by AAAAAAAAA.