Auxiliary Factors and RNA Substrates Regulate Dead-Box Protein Activity by Modulation of the Dead-Box Protein Conformational Cycle

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DEAD-box proteins unwind RNA duplexes at the expense of ATP hydrolysis in virtually all processes involving RNA. During unwinding, they alternate between open and closed conformations, and the transition to the closed conformation has previously been linked to duplex destabilization. The eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box helicase that is thought to resolve secondary structure elements from the 5′-UTR of mRNAs to enable ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. eIF4A can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by eIF4G, and a closed state populated in the presence of eIF4G and eIF4B. In single molecule FRET experiments on donor/acceptor-labeled eIF4A, we have dissected the effect of eIF4B and eIF4G on RNA-dependent ATPase- and RNA helicase activities, and on the eIF4A conformational cycle in the context of different unwinding substrates. We show that eIF4B and eIF4G, as well as different structures in the 5′-UTR, modulate the energy landscape underlying the eIF4A conformational cycle by changing the energetic differences and the energy barriers between functionally relevant conformational states, leading to changes in rate constants for inter-conversion and in equilibrium distributions. Our results reveal on a molecular level how translation initiation factors synergistically stimulate the eIF4A helicase activity in the mRNA scanning process, and show that the eIF4A conformational cycle is central for the multi-layered regulation of eIF4A activity, and for its role as a regulatory hub in translation initiation.