Large ribosomal subunit, eIF5B, Met-tRNAiMetand mRNA cooperate to complete accurate initiation

Recognition of a start codon by the first aminoacyl-tRNA (Met-tRNA i Met ) 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 i Met 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 i Met 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. One sentence summary CryoEM structure of a naturally long-lived translation initiation intermediate with Met-tRNA i Met and eIF5B post GTP hydrolysis.