Eukaryotic initiation factor eIF3 facilitates loading of eukaryotic release factor eRF1 or suppressor tRNA to the ribosome.

Eukaryotic translation initiation factor eIF3 plays a pivotal role in 48S preinitiation complex assembly and ribosomal scanning. It binds simultaneously to the 40S ribosomal subunit and the eIF4F cap-binding complex, which, through its interaction with poly(A)-binding protein (PABP), facilitates closed-loop mRNA structure formation. PABP also interacts with the eukaryotic release factor eRF3, thereby co-localizing initiation and release factors, and suggesting potential functional crosstalk. Using a reconstituted mammalian translation system, we demonstrate that eIF3 significantly enhances translation termination. Specifically, eIF3 promotes the loading of eRF1 into the ribosomal A site, accelerating the GTPase activity of eRF3 and the rate of peptide release. We also show that eIF3 facilitates the binding of suppressor or near-cognate tRNAs to stop codons to enable readthrough and continued elongation. These findings establish a conserved, direct role of eIF3 in regulation both translation termination and stop codon readthrough, a mechanism particularly relevant within closed-loop mRNA structures and during upstream open reading frame translation.