6200 Nucleic Acids Research, 2012, Vol. 40, No. 13 eIF4A is the prototypical member of the DEA(D/H)- box RNA helicase family, and is one of the more abundant translation initiation factors, present at 3 copies/ribosome (17). eIF4A is present as two isoforms, eIF4AI and eIF4AII, which share 90–95% sequence identity and are thought to be functionally interchange- able (18). The helicase activity of eIF4AI is signiﬁcantly stimulated by the presence of the translation initiation factors, eIF4B or eIF4H, or when eIF4A is present as a subunit of the eIF4F complex (eIF4Ac) (8,19–21). Although eIF4AI has been extensively studied using classical biochemistry approaches (19,22,23), the mechan- isms underlying its ligand-dependent conformational changes, its helicase activity as an individual unit, and its signiﬁcant activity enhancement in the presence of eIF4B or eIF4H, have all to date remained incompletely understood. Moreover, a direct and unambiguous meas- urement of eIF4AI’s position on the mRNA template during RNA unwinding has not been reported. Bulk studies of eIF4AI helicase activity have classically used gel electrophoresis oligonucleotide displacement assays, which only detect the completely unwound products and have not provided insight into unwinding dynamics. In contrast, sm techniques, in particular, ﬂuorescence resonance energy transfer (sm-FRET), can provide direct and highly quantitative accounts of eIF4AI location and re-positioning on its RNA substrate during unwinding, and is well suited to eIF4AI’s mechanism of action. This technique’s utility for understanding helicase activity has been highlighted by recent studies employing sm-FRET to determine the spring-loaded DNA unwinding mechanism of Hepatitis C NS3 helicase (24) and to probe a fast repetitive motion of Escherichia Coli Rep helicase (25). Here, we employ sm-FRET to probe eIF4AI’s unwinding activity. We ﬁnd that eIF4AI alone can induce rare and isolated unwinding episodes of RNA duplexes. We also use sm-FRET to probe the consequences of eIF4H, another translation initiation factor shown to interact with eIF4AI (26), on the unwinding parameters of eIF4AI and ﬁnd that the resulting eIF4AI/eIF4H complex exhibits an abundance of persistent, repetitive cycles of unwinding over extended periods of time. We ﬁnd that eIF4AI is directed to bind to single-stranded loop structures by eIF4H, facilitating its ability to repeti- tively unwind duplexes in an ATP-dependent manner. MATERIALS AND METHODS RNA/DNA substrate preparation DNA and RNA oligonucleotides were purchased from IDT (Coralville, IA, USA) and Dharmacon (Lafayette, CO, USA), respectively. The oligonucleotides were labeled using Cy3 or Cy5 (GE Healthcare, Little Chalfont, UK) according to the vendor’s recommended protocol. Oligonucleotides and dyes were mixed at a molar ratio of 1:15, respectively. The conjugation included incubating samples overnight in 0.1 M sodium tetraborate buffer (pH 8.5) at room temperature. Conjugation was conﬁrmed using native polyacryl- amide gel electrophoresis (PAGE) and quantiﬁed by UV–Vis absorption. RNA/DNA hybrid duplexes were formed by annealing the oligonucleotides in 50 mM Tris–HCl (pH 7.5), 0.2 mM EDTA and 500 mM NaCl, heating the samples to 95 C, and slowly cooling them to 4 C over a period of 2.5 h. The duplexes were then puriﬁed using native PAGE. Hybrid hairpin structures were prepared by mixing DNA oligonucleotides, which contained a loop region, with single-stranded RNAs. The DNA oligonucleotides contained a terminal biotin moiety used for surface immo- bilization in the sm assays. Ligation of the DNA/RNA strands was performed with T4 RNA ligase 2 (New England Biolabs, Ipswich, MA, USA), following which substrates were puriﬁed by denaturing PAGE to remove the unligated oligonucleotides. The preparation of a blunt-ended hairpin substrate was performed by mixing the puriﬁed hairpin sample with an additional oligo- nucleotide (23 nt ssDNA) at a hairpin:ssDNA molar ratio of 1:10, heating to 95 C, and allowing to slowly cool to 4 C over a period of 2.5 h. The blunt-end hairpin was puriﬁed using native PAGE. The sequences of all oligonucleotides used in this study are shown in the Supplementary Figure S1. Protein expression and puriﬁcation Recombinant His6-eIF4AI and His6-eIF4H were expressed in E. coli BL21-Gold (DE3) cells (Stratagene, La Jolla, CA, USA). Following IPTG-induction of expres- sion, lysis of cells by sonication and clearing of the supernatant by centrifugation, His6-eIF4AI-containing lysate was loaded on a Ni++-Sepharose 6 Fast Flow resin (GE Healthcare, Piscataway, NJ, USA). The matrix was washed, eluted with A300 buffer [20 mM Tris (pH 7.5), 10% glycerol, 300 mM KCl, 0.1 mM EDTA] containing 0.2 M imidazole. Following dialysis against A100 buffer [20 mM Tris (pH 7.5), 10% glycerol, 100 mM KCl, 0.1 mM EDTA and 2 mM DTT], eIF4AI was loaded onto a Q-Sepharose fast ﬂow matrix (GE Healthcare). The resin was washed with 10 column volumes of A100 and protein eluted using a linear salt gradient from 100 mM to 500 mM KCl in buffer A (20 mM Tris (pH 7.5), 10% glycerol, 0.1 mM EDTA, 2 mM DTT). The eluted fractions were analyzed by SDS–PAGE and those containing eIF4AI were pooled and dialyzed against buffer A. His6-eIF4H was ﬁrst enriched on a Ni++-Sepharose column then further puriﬁed on a Heparin-Sepharose matrix (GE Healthcare). After applying the sample, the resin was washed with 1 column volume of A100 containing 2 mM DTT, and eIF4H eluted with a linear salt gradient from 100 to 500 mM KCl in buffer B [20 mM Tris (pH 7.5), 10% glycerol, 0.1 mM EDTA and 2 mM DTT]. The eluted fractions were analyzed by SDS–PAGE and those containing t His6-eIF4H were pooled and dialyzed against storage buffer [20 mM HEPES (pH 7.5), 25% glycerol, 50 mM KCl, 0.1 mM EDTA and 1 mM DTT]. Bulk FRET assay In the bulk measurements, the donor (Cy3) and the acceptor (Cy5) signals were collected using a home-build