A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation

Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during transla-tion initiation. However, the mechanism of its cou-pled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release con-trolled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA un-winding. Molecular dynamic simulations and exper-imental results revealed that, through forming a hy-drophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal do-main, the linker gated the release of Pi from the hy-drolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases