First, a stable formation of a 43S PIC on the 5′UTRPab1 was confirmed by an electrophoresis mobility shift assay (EMSA) (Supplemental Fig. S1A). Next, the 5′UTR alone and 5′UTRs with 40S and initiation factors assembled were immobilized onto the streptavidin-coated magnetic beads and were incubated with yeast cell extracts. Following an extensive wash to remove most of the material bound nonspecifically, proteins that bind to the 5′UTR were eluted with D-biotin and analyzed by mass spectrometry. Using this assay, we confirmed that Sbp1 specifically binds to 5′UTRPab1 (Supplemental Fig. S1B). At the same time, we also pulled down Pab1 protein from the cell extract using this assay (Supplemental Fig. S1B, lanes 3,5, and 7). This result is in good agreement with earlier findings that Pab1 binds to the polyadenosine sequences in its own 5′UTR (Sachs et al. 1987).