Large ribosomal subunit, eIF5B, Met-tRNA i Met and mRNA cooperate to complete accurate initiation

Recognition of a start codon by the first aminoacyl-tRNA (Met-tRNA) determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of Met-tRNA on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determined the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNA immediately before transition into elongation. The structure uncovered an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.