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•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

Laboratory of Gene Regulation and Development Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda United States

Laboratory of Regulation of Gene Expression Institute of Microbiology ASCR Prague Czech Republic

Laboratory on the Mechanism and Regulation of Protein Synthesis Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda United States

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uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Please do not recycle! Translation reinitiation in microbes and higher eukaryotes

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle

In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation