Kinetics of mRNA Recruitment to PICs

The kinetics with which a capped rpl41a mRNA was recruited to PICs was determined using a previously described native gel electrophoresis-based assay to monitor the mobility of a [³²P]-cap-labeled rpl41a mRNA^{22 (link),36 (link),53 (link),54 (link)}. A 2× PIC Master Mix was formed by combining 2 µM eIF1, 2 µM eIF1A, 600 nM eIF2, 750 nM Met-tRNA^iMet, 4 µM eIF4A, 800 nM eIF4B, 100 nM 40S subunit, and 4 mM ATP•Mg²⁺. This 2× PIC Master Mix was then used to generate a 1× reaction by adding stock solutions of eIF3_R or eIF3_N to the final specified concentrations. mRNA recruitment reactions were initiated by adding [³²P]-cap-labeled rpl41a mRNA to a final concentration of 15 nM. mRNA recruitment reactions were incubated continuously at 26 °C. At each specified time point, 4 µL of the reaction was removed and combined with 1 µL of Native Loading Dye and loaded onto an actively running 4 % polyacrylamide (37.5:1 acrylamide:bis-acrylamide) gel prepared in THEM Buffer. This gel was then run for 70 min at 200 Volts, and maintained at 16 °C with a circulating water cooler^{22 (link),37} . Subsequently, the gel was exposed to a phosphor screen overnight and then imaged on an Amersham Typhoon 5 Biomolecular Imager (Cytiva, Catalog Number 29187191).