Structural basis for non-AUG translation regulation by 5MPs

The cellular proteome is regulated by translation initiation on AUG or non-canonical (non-AUG) start codons. Non-AUG initiation remodels proteome during stress and is implicated in cancer and other diseases. The eIF5-mimic proteins (5MPs) restrict non-AUG start codon usage and thereby reprogram proteoform expression from mRNAs with alternative start sites, such as the oncogenic c-Myc. The mechanism by which 5MPs induce such translational reprogramming remains unknown. Here, using in extracto cryo-electron microscopy (cryo-EM) and biochemical assays, we report that translational repression by 5MP strongly depends on the sequence context near the AUG or non-AUG codons. Cryo-EM structures of 5MP-bound 48S pre-initiation complexes (PICs) from native cell extracts reveal that 5MP binds at the A site of the small ribosomal subunit, stabilizing an expanded open-head conformation of the PIC scanning along mRNA. The N-terminal region of 5MP blocks the A site, whereas the C-terminal domain docks at eIF2β and the initiator tRNAMet outside the P site (i.e., Pout). These findings indicate that 5MP protein directly biases the initiating 48S complexes toward the open conformation, promoting mRNA scanning and inhibiting initiation at suboptimal start codons.