6204 Nucleic Acids Research, 2012, Vol. 40, No. 13 and ‘unwinding time’ (low FRET level). Statistical analysis of thousands of these transitions revealed expo- nential distributions of the dwell times for each state, where the characteristic unwinding time was found to be U = 0.71 ± 0.02 s and the characteristic waiting time was found to be W = 1.35 ± 0.05 s (Figure 3C). To exclude the possibility that the observed FRET tran- sitions stem from binding/unbinding events of multiple eIF4AI/eIF4H complexes to the nucleic acid substrates, we documented FRET activity (in the presence of eIF4AI, eIF4H and ATP) for a set of substrate molecules, followed by ﬂushing the ﬂow cell with more than 5 volumes of buffer lacking eIF4AI, eIF4H or ATP and monitoring FRET activity for 60 s (Figure 4). This treatment, which resulted in a loss of FRET ﬂuctuations (Figure 4B), was then followed by re-introduction of only ATP into the system. We noted a resumption of repetitive FRET ﬂuctu- ations (Figure 4C) for the majority of the observed sm (some molecules were either photo-bleached during the process or washed away). We interpret these results to indicate that the FRET activity observed post-ATP re- introduction is due to the presence of eIF4AI/eIF4H proteins that have remained bound to the duplex strand during all washes. Taken together with the results above illustrating that the observed repetitive FRET transitions are ATP-dependent and reﬂect a change in donor–acceptor distance of 3 nm, they most likely result from an oscilla- tory unwinding/reannealing pattern of the same eIF4AI/ eIF4H complex that remained bound to its substrate. The eIF4AI/eIF4H complex preferably binds to the loop region of hairpins To further evaluate the stimulatory role of eIF4H on eIF4AI unwinding dynamics, we explored how substrate design could affect eIF4AI activity. It has been reported that eIF4AI unwinds RNA substrates with single-strand overhangs slightly more efﬁciently than blunt ended RNA duplexes (30). By analogy, we speculated that eIF4H facilitates duplex unwinding activity of eIF4AI by binding ﬁrst to single-stranded portions of the RNA. This would serve two goals, both increasing the afﬁnity of the eIF4AI/eIF4H complex for single-stranded domains of the RNA transcript as compared to the afﬁnity of eIF4AI alone for such substrates, and also allowing for a more processive unwinding mechanism. In order to explore the binding afﬁnity of eIF4AI to the RNA substrate in the absence or presence of eIF4H, we performed a series of electrophoretic mobility shift assays (Supplementary Figure S5). A distinct complex is not apparent upon incubation of eIF4AI or eIF4H with RNA substrate. However, both proteins together are capable of forming a distinct complex in the presence of ATP or ATP- S (Supplementary Figure S5, lanes 7 and 8). Furthermore, we found that eIF4AI and eIF4H formed more stable complexes when the loop size of the hairpin substrates exceeded 6 nt (Supplementary Figure S6). The preferred binding location of the eIF4AI/eIF4H complex to the RNA substrates used in these experiments was determined by two additional sm-FRET experiments. First, a complementary DNA oligonucleotide located adjacent to the biotin residue was used to block the single-stranded portion of the RNA substrate (Supple- mentary Figure S7). For these ‘blocked’ samples, repeti- tive FRET ﬂuctuations were observed and persisted to the same extent as reported above, indicating that a single-stranded overhang does not contribute signiﬁcantly to eIF4AI/eIF4H’s repetitive unwinding capability. Next, under the assumption that that eIF4H must preferentially bind to the loop region, we tested substrates with different loop sizes, ranging from 3 to 12 nt long. In each of these experiments, we collected at least 1500 sm traces and Figure 4. eIF4AI/eIF4H complex remains bound to the hairpin substrate during cycles of repetitive FRET signal oscillations. A typical FRET trace for a single substrate molecule acquired in reactions containing: (A) both eIF4AI, eIF4H (1 mM) and ATP (1 mM); (B) after>5 volume ﬂushing of the chamber with buffer lacking eIF4AI, eIF4H and ATP (‘Wash 1’); (C) following reintroduction of only ATP at 1 mM (‘Wash 2’).