We cloned 5′UTRPab1 and its sequence complement, 5′UTRPab1-complement, as PCR-generated BamHI–HindIII fragments into the pTRAPv5 vector (Cytostore Inc.) downstream from the two streptavidin-binding S1 aptamers (Srisawat and Engelke 2001). Vectors were linearized with HindIII. Uncapped and aptamer-tagged RNA were transcribed from the DNA templates using T7 polymerase and purified by denaturing polyacrylamide gel electrophoresis or anion-exchange chromatography (Easton et al. 2010). The bait complex that we used in this assay corresponds to the 43S pre-initiation complex (43S PIC) that consists of a ternary complex (eIF2•GTP•Met-tRNAiMet) and a 40S ribosomal subunit (Jackson et al. 2010). Since binding of eIF1 and eIF1A to the 40S ribosome facilitates the recruitment of the ternary complex (Lorsch and Dever 2010), we also included eIF1 and 1A in the bait. Furthermore, we used the nonhydrolyzable GTP analog GDPCP to stabilize the ternary complex in the GTP state.