Dynamics and Regulation of mRNA Cap Recognition by Human eIF4F

SUMMARY Efficient eukaryotic messenger RNA translation requires dynamic collaboration between the three subunits of initiation factor 4F (eIF4F, eIF4E•G•A), which recognises and activates mRNA at its 5ʹ cap structure for ribosome recruitment. Despite its high biological and pharmacological importance, the dynamics of full human eIF4F–mRNA engagement remain largely uncharacterised, hindering mechanistic understanding of translation initiation and its regulation. Here we observed human eIF4F activity with single-molecule fluorescence assays that directly visualise mRNA cap recognition by its eIF4E subunit. Unexpectedly, we find that inherently transient eIF4E–cap binding is repressed by full-length human eIF4G, predominantly through its C-terminus, representing an unanticipated role for eIF4G as a central rate-limiting factor in the eIF4F complex. This repression is relieved by nucleotide-bound eIF4A in the eIF4F heterotrimer, placing eIF4A as a crucial determinant of efficient cap recognition for translation. Molecular dynamics simulations reveal that electrostatic modulation of eIF4E–mRNA interaction allows eIF4G to control the cap-recognition frequency. Our findings also indicate that intrinsic eIF4F– mRNA dynamics are insufficient to support cap-tethered ribosomal scanning to locate translation start sites. They illuminate fundamental design-principle differences for the overall mechanism and division of labour among eIF4F subunits during mRNA recognition in humans and yeast. Competing Interest Statement The authors have declared no competing interest. DATA AVAILABILITY All data analysed in this study are available in the manuscript and Extended Data.