Linking translation factor conformational dynamics with translation efficiencies and gene expression.

Abstract

Translation initiation is a tightly regulated step in eukaryotic translation; deregulation leads to altered gene expression, tumor formation and cancer. The process involves a large set of initiation factors (eIFs), including the DEAD-box helicase eIF4A, an ATP-dependent RNA helicase responsible for unwinding of secondary structures in the 5’-UTR of mRNAs. Together with the cap-binding protein eIF4E and the scaffold protein eIF4G, eIF4A forms the eIF4F complex that recognizes the 5’-cap and, assisted by eIF4B, recruits the 40S ribosomal subunit. The resulting pre-initiation complex then scans through the 5’-untranslated region (5’-UTR) towards the start codon, where protein synthesis commences. Here, we investigate the regulation of eIF4A activity by other translation factors and by the 5’-UTR. In single-molecule FRET experiments in solution and on surface-immobilized eIF4A we show that eIF4B and eIF4G modulate eIF4A activities by accelerating the conformational changes that are coupled to ATP hydrolysis and RNA unwinding. We further dissect the intricate interplay of individual domains of eIF4G, and the interaction with eIF4E and the 5’-cap, and show that eIF4A conformational dynamics and activities are fine-tuned through a delicately balanced network of stimulatory and inhibitory effects. Strikingly, the 5’-UTR itself also modulates eIF4A activity by altering its conformational dynamics. 5’-UTRs of mRNAs with different eIF4A dependence in vivo achieve different levels of eIF4A stimulation, suggesting that a link between the conformational dynamics of eIF4A and eIF4A dependence. By combining single-molecule FRET experiments with in vitro translation assays, we provide evidence that the conformational dynamics of eIF4A is a key determinant for eIF4A dependence, and for the efficiency of translation initiation and gene expression.