6206 Nucleic Acids Research, 2012, Vol. 40, No. 13 irreversibly terminates the FRET oscillations since these require a hydrolytic event. DISCUSSION Sm approaches can be used to directly quantify a myriad of biomolecular processes, providing observational access to, and information about, their detailed dynamics under physiological conditions, especially when used in combin- ation with classical biochemical assays. Here, we utilized sm-FRET to probe the unwinding dynamics of eukaryotic initiation factor eIF4AI in the presence of the RNA- binding factor eIF4H. Our results indicate that the assembly of the eIF4AI/eIF4H complex on the RNA tran- script, and subsequent unwinding of its duplex regions can be signiﬁcantly enhanced by long-lived binding of the complex to single-stranded loop domains longer than a critical minimum length of 6 nt. In accordance with previous studies indicating that eIF4H binds to RNA via the RNA recognition motif (RRM) domain, our results support a simple RNA unwinding model, where repetitive zipper-like unwinding/re-annealing cycles can be efﬁciently executed by the eIF4AI/eIF4H complex when bound to a loop domain within the secondary struc- ture. Upon ATP hydrolysis, unwinding of the duplex region induces rapid high-to-low FRET level transitions (Figures 3 and 4). This mode of short distance helicase activity is quite efﬁcient, consuming a single ATP molecule per cycle of unwinding. The unwinding process can be terminated upon binding of the non-hydrolysable ATP analog, ATP- S. The ATP dependence of the unwinding activity of eIF4AI/eIF4H is reminiscent of other DEAD-box helicases such as CYT-19 and DbpA, which have been shown to achieve complete strand separ- ation of RNA duplexes via hydrolysis of a single ATP molecule (37–39). Linking eIF4AI activity to eIF4H function establishes a crucial means by which eIF4AI activity could be regulated. Generally, eIF4H activity involves directing other cellular proteins to speciﬁc mRNA structures to enhance their activity. For example, eIF4H binding is required for the herpes simplex virus virion host shutoff (Vhs) protein-induced degradation of many mRNAs, where it targets Vhs to preferred sites within mRNAs (40). siRNA-mediated depletion of eIF4H, but not eIF4B, in HeLa cells impedes Vhs-mediated mRNA deg- radation (40). We ﬁnd that eIF4H plays a similar role in stimulating eIF4AI activity. The marked increase in unwinding activity observed for hairpin substrates, as compared to a simple duplex with an overhang, suggests that the eIF4AI/eIF4H complex binds to hairpin loops with higher afﬁnity than to single-stranded overhangs. This conﬁguration allows it to maintain close interaction with both strands in order to initiate multiple unwinding episodes, which in turn keeps the RNA in its unwound state for a longer total period of time. Another conceivable advantage of this conﬁguration is that the eIF4AI/eIF4H repeated unwinding activity augments downstream steps, ultimately and indirectly stimulating ribosome binding, and consequently protein synthesis. eIF4H, like eIF4B, enhances the helicase activity of eIF4A and contains an RRM that shows sequence homology to eIF4B in this region—however, the two proteins are dissimi- lar in their mechanism of action and their inﬂuence on eIF4A RNA binding and ATPase activity (20). It remains to be determined whether our results with eIF4H can be extrapolated to eIF4B and will exhibit a complementary function in our assay. The mRNA 50-UTRs present a complex landscape of short stem and loop regions. Our data suggests that if eIF4H could be delivered internally to such loops, in conjunction with eIF4AI, RNA remodel- ing should rapidly occur. This might play an important role in IRES-dependent translation initiation. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online: Supplementary Figures 1–10 and Supplementary References [41,42]. FUNDING Human Frontiers Science Program (HFSP) award RGP0036-2005 (to A.M. and N.S.); Canadian Institutes of Health Research (MOP-106530); Ministere du D e´veloppement Economique, Innovation et Exportation (to J.P.). Funding for open access charge: HFSP award [RGP0036-2005]. Conﬂict of interest statement. None declared. REFERENCES 1. Sonenberg,N. and Hinnebusch,A.G. (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell, 136, 731–745. 2. Sonenberg,N. (1996) mRNA 50 cap-binding protein elF4E and control of cell growth. In: Hershey,J.W.B., Matthews,M.B. and Sonenberg,N. 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