eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.