Reinitiation and Other Unconventional Posttermination Events during Eukaryotic Translation

• eIF2, eIF3, eIF1, and eIF1A promote bidirectional reinitiation by recycled 40S subunits
• Imposition of 3′ directionality on reinitiation additionally requires eIF4F
• Posttermination 80S ribosomes are mobile and can migrate to cognate codons
• eEF2 induces dissociation of eRF1 and promotes 80S ribosomal migration

SummaryDuring ribosome recycling, posttermination complexes are dissociated by ABCE1 and eRF1 into 60S and tRNA/mRNA-associated 40S subunits, after which tRNA and mRNA are released by eIF1/eIF1A, Ligatin, or MCT-1/DENR. Occasionally, 40S subunits remain associated with mRNA and reinitiate at nearby AUGs. We recapitulated reinitiation using a reconstituted mammalian translation system. The presence of eIF2, eIF3, eIF1, eIF1A, and Met-tRNAiMet was sufficient for recycled 40S subunits to remain on mRNA, scan bidirectionally, and reinitiate at upstream and downstream AUGs if mRNA regions flanking the stop codon were unstructured. Imposition of 3′ directionality additionally required eIF4F. Strikingly, posttermination ribosomes were not stably anchored on mRNA and migrated bidirectionally to codons cognate to the P site tRNA. Migration depended on the mode of peptide release (puromycin > eRF1⋅eRF3) and nature of tRNA and was enhanced by eEF2. The mobility of posttermination ribosomes suggests that some reinitiation events could involve 80S ribosomes rather than 40S subunits.