calculated the fraction of traces displaying the oscillating FRET pattern in each experiment. The results indicate that FRET oscillations are practically non-existent when the loop size is shorter than 6 nt (Figure 5). Furthermore, the FRET assays showed a distinct increase in the percent of substrate molecules undergoing oscillations for longer loop sizes, reaching 65% when the loop size is 12 nt long. The results suggest that the critical minimum loop size required for the eIF4AI/eIF4H complex to bind and initiate repetitive unwinding activity upon an RNA substrate is 6 nt. Sm measurements suggest that the repetitive RNA unwinding involves a single ATP hydrolysis per FRET cycle To explore the role of ATP hydrolysis in the repetitive unwinding process, we reduced the ATP concentration from 1 mM down to 30 mM and measured the resulting sm-FRET dynamics (Figure 6A). If the ‘waiting time’ tw, represents the time required for the recruitment of a fresh ATP molecule from bulk, then reducing ATP concentration to the point where ATP diffusion time becomes the rate-limiting step is expected to affect tw but not ‘unwinding time’, U. This prediction is conﬁrmed by our data. A 60% increase in tw (circles) was observed upon reduction of the ATP concentration below 0.1 mM, whereas the unwinding time (triangles) remained unaffected ( U = 0.64 ± 0.15 s) for all ATP concentrations measured (the dwell-time distributions for each data point are shown in Supple- mentary Figure S8). Moreover, the fact that U does not increase upon a decreasing the ATP concentration, while tw does increase, suggests that a single ATP molecule is consumed per unwinding cycle since consumption of two or more ATP molecules per cycle would have resulted in a concurrent increase in U when the ATP concentration falls below 0.1 mM. We also tested whether or not the non-hydrolysable ATP analog, ATP- S, could substitute for ATP in the Figure 5. eIF4AI/eIF4H complex requires hairpin substrates contain- ing minimum loop sizes of 6 nt for repetitive unwinding. The fraction of sm traces showing > 3 oscillations in a 60 s monitoring time window is plotted as a function of loop length (in nucleotides). Each data point consists of at least 1500 sm traces. Nucleic Acids Research, 2012, Vol. 40, No. 13 6205 Figure 6. Dependence of the hairpin substrate unwinding dynamics by the eIF4AI/eIF4H complex on ATP or ATP-gS concentrations. In these experiments, 12-nt hairpin loop structures were used, and 1 mM of both eIF4AI and eIF4H. (A) Relationship of the waiting times ( W, circles) and the unwinding times ( U, triangles) on the ATP concentration. Each data point represents statistics of at least 1000 sm traces (detailed distributions are provided in the Supporting Information ﬁle). (B) The dependence of waiting times ( W, circles) and unwinding times ( U, triangles) on the relative ATP-gS concentration, with a ﬁxed ATP concentration of 0.1 mM. The abscissa x ¼ ½ATP S =ð½ATP +½ATP SÞ . Each data point represents statistics of at least 500 sm traces. unwinding reaction (Figure 6B). By keeping the total concentration of [ATP]+[ATP- S] ﬁxed, we found that increasing the fractional amount of ATP- S up to 15% had little effect on the waiting time or the unwinding time (the dwell-time distributions for each data point are shown in Supplementary Figure S9). However, we ﬁnd that both the fraction of sm traces showing consecutive sm-FRET oscillations within a ﬁxed 60 s monitoring interval, as well as the average number of repetitive FRET cycles observed for any given molecule, displayed a 3- to 4-fold decrease with increasing x values (Supplementary Figure S10). This experiment suggests that ATP- S can substituted for ATP in the initial eIF4AI/eIF4H complex formation, but