Since Pab1 binds to the poly(A) region in the 5′UTRPab1 (Sachs et al. 1987) and both proteins are pulled down simultaneously on the 5′UTRPab1 with the 43S PIC in our TRAP assay (Supplemental Fig. S1B), these results suggest that Sbp1 and Pab1 likely bind to the 5′UTRPab1 at the same time. Indeed, a higher-order complex of 5′UTRPab1–Sbp1–Pab1 is formed under our experimental conditions, as shown in a native EMSA (Fig. 2C).