eIF3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2{middle dot}GTP{middle dot}Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we un