The translation initiation factor DHX29 appears to pull on mRNA in a direction opposite to scanning.

The DExH-box helicase DHX29 plays a critical role in mammalian translation initiation. It is required for the scanning of mRNAs with complex 5UTRs. Despite its importance, the detailed mechanism of DHX29s action has remained debated. Using structural models derived from AlphaFold and cryo-EM structure of DHX29 bound to the ribosomal 43S pre-initiation complex, we provide a revised structural framework that clarifies the interplay between DHX29, the 40S ribosomal subunit, and eIF3. Our findings suggest that the 40S subunit regulates DHX29s NTPase activity through an activation mechanism resembling the G-patch protein regulation of DEAH helicases. Moreover, our model supports a 3 to 5 translocase mechanism, in which DHX29 transiently pulls the mRNA opposite to the scanning direction, destabilizing stable stem-loops trapped in the mRNA channel and allowing scanning to resume. This structural analysis refines our understanding of DHX29s function and provides new hypotheses regarding its role in mRNA unwinding during scanning and start codon selection.