biostudies-literatureLapointe CPNIA NIH HHSNIGMS NIH HHSNIH HHS185-190https://www.ebi.ac.uk/biostudies/studies/S-EPMC9728550biostudies-literatureUnknown607(7917)Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases^1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. 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 the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-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 translation initiation in humans.NatureeIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.PMC9728550R01 GM113078R01 GM092927K99 GM144678RF1 AG064690AG064690R35 GM145306R01 AG064690R00 GM144678R01 GM051266GM011378Shin BSPuglisi JDSokabe MLapointe CPMontabana EVilla NGrosely RAlvarado CDever TEFraser CSFernandez ISWang JfalseeIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases^1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. 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 the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-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 translation initiation in humans.2022-01-01T00:00:00Z2022 Jul2026-05-28T12:55:00.308Z2025-02-18T22:34:41.531ZS-EPMC97285503573273510.1038/s41586-022-04858-z