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Cheng et al. 10.1073/pnas.0702315104.

Supporting Information

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Fig. 7. NS3 monomer has ATP-dependent RNA helicase activity at zero force. Fraction of substrates unwound as a function of [NS3] in a bulk single-cycle unwinding assay. Different points at a single [NS3] correspond to different experiments. The RNA substrate is a 10-bp duplex (80% GC content) with a 10-nt 3'-ssRNA overhang and gP32 label at the 5' end of the short strand. NS3 at varied concentrations (20-100 nM) is incubated with 100 nM substrate for 3 min at 22°C and then mixed with 1 mM ATP, 2 mM NS3 trap (60 nt) [Pang PS, Jankowsky E, Planet PJ, Pyle AM (2002) EMBO J 21:1168-1176], and 4 mM trap for the short RNA strand (a DNA oligonucleotide with 10-bp complementarity to the short RNA strand, necessary to capture the unwound RNA and prevent its reannealing). An incubation time longer than 3 min does not change the result, which is also in contrast to the long incubation time (typically 1 h) to observe NS3 dimer activity. The NS3 trap concentration is sufficient under these conditions because no unwinding is observed after preincubating NS3 at its highest concentration used (100 nM) with both RNA substrate and NS3 trap. All concentrations reported are final concentrations after mixing. The unwinding reactions lasted 5 min at 22°C and were then stopped by addition of an equal volume of 0.3 M EDTA plus 20% vol/vol glycerol. The resulting solution was mixed vigorously before loading onto a 20% native PAGE gel to separate unwound product from intact substrate. The gel was run in a 4°C room and quantified with autoradiography. Although the unwinding amplitude is low (6-7%), the dependence of unwinding amplitudes on [NS3] suggests that unwinding is catalyzed by a monomer of NS3, as indicated by the fit to a model where NS3 monomer has helicase activity (red solid line). Conversely, a model where only NS3 dimer has helicase activity does not describe the data (blue solid line). In both fitting, the equilibrium constant for NS3 monomer binding to RNA is set to 6 nM and the equilibrium constant for NS3 dimerization on RNA is 19 nM [Serebrov V, Pyle AM (2004) Nature 430:476-480]. The data points for NS3 concentration were stopped at 100 nM simply due to the incompatibility of high NaCl concentration in NS3 storage buffer with the working solution for helicase assay that contains only 30 mM NaCl. Controls without ATP show no unwinding at the highest NS3 concentration used.

Fig. 8. Dependence of helicase mean stepping velocity on the free energy of base pair opening. For all panels, the mean stepping velocity of NS3 (blue symbols) depends less on the free energy of base pair opening than that of a passive helicase (dashed lines). The y-intercepts of the dashed lines are obtained from the y-intercepts of straight lines (not shown) that fit NS3 stepping velocity. A, B, and C are for openings of 2, 3, and 5 bp, respectively.

Fig. 9. Dependence of NS3 processivity on sequence barriers in RNA. (A) Sequence comparison among substrates RNA-AG, RNA3, and RNA-GA. (B) The fraction of unwinding is plotted as a function of the length of duplex RNA unwound by NS3 (cumulative processivity plot) for RNA-AG (9 pN, 44 traces), RNA3 (9 pN, 69 traces), and RNA-GA (11 pN, 31 traces), respectively. The area underneath each curve represents the average number of base pairs that can be unwound by NS3 before dissociation from that particular substrate, which is a measure of the average processivity of the helicase. On average, only 12 bp of G•C are unwound at 11 pN for RNA-GA before enzyme detachment. The slight negative slope of NS3 processivity on A•U portion of RNA-AG indicates that, on average, 150 bp of A•U is unwound by NS3 before dissociation. The error bars are standard deviations calculated from bootstrap analysis for the same data sets sampled 1,000 times.

Fig. 10. Characterization of RNA-6GC and RNA-3GC. (A) Force-extension curves of RNA-6GC unfolding (olive) and refolding (orange) by mechanical force. Note the 2^nd transition near 20 pN. (B) Force-extension curves of RNA-3GC unfolding (green) and refolding (orange) by mechanical force. Note the second transition near 17 pN. The mechanical unfolding and refolding speeds are 200 nm/s for A and B.

Fig. 11. Dependence of RNA hairpin unfolding free energy on salt concentration. The free energies of RNA3 hairpin unfolding were determined by performing mechanical unfolding experiments at various [NaCl] in 20 mM Mops, 0.9% vol/vol glycerol, 0.75 mM MgCl₂, 0.1% Tween 20, 2 mM DTT, and 1 mM ATP (pH 6.5) (10). The error bars for each data point are the standard deviations of the free energy values calculated from 30 repeats of the same experiment. The dependence of the free energy of unfolding on log[NaCl] is fitted to a straight line (red solid line; R² = 0.91) that gives a correction factor of 73% in relating the free energy of base pair opening at 1 M NaCl to that at 30 mM NaCl.

Table 2. Independence of NS3 unwinding kinetics on external force held on the substrate

Data from RNA-AG were analyzed for pause duration and stepping velocity on 100% A•U at three different forces. Numbers of traces included in the analysis were 40, 96, and 35 for 5, 7, and 9 pN force, respectively. The independence of NS3 pause duration or stepping velocity on external force applied to the substrate suggests that pause and stepping are intrinsic to the enzymatic cycle of the helicase and further supports our conclusion that NS3 uses an active mechanism to unwind RNA. Otherwise, both pause duration and stepping velocity should be force-dependent as one would expect for a purely passive Brownian ratchet.

Table 3. The free energy of RNA base pair opening and its reduction by NS3

The size of NS3 unwinding substeps ranges from 2 to 5 bp as observed previously (1) on a hairpin sequence with 52% G•C content. Here, we consider free energy values of base pair opening ranging from 1 to 5 bp (first column). These values are listed in the order of 100% A•U, 52% G•C, and 100% G•C within each cell on the second column and correspond to 30 mM NaCl at 22°C. The free energy of base pair opening was first calculated by using Mfold 2.3 (2) at 22°C. Because Mfold energy parameters are for 1 M NaCl without Mg²⁺, we performed mechanical pulling experiments for RNA3 in buffer U supplemented with increasing concentrations of NaCl to obtain free energy of unfolding (3) at different [NaCl]. The free energies of unfolding at various [NaCl] were well described by a linear relationship (SI Fig. 11), which yielded a correction factor of 73% in relating free energy at 30 mM NaCl to 1 M NaCl. These salt titration experiments were also performed in the presence of 1 mM ATP, which was the ATP concentration used for all the unwinding experiments reported in this article. Because our condition is [ATP]>[Mg²⁺], we expect that [Mg²⁺] has a minimum effect on duplex RNA stability. The mean stepping velocity of NS3 (Table 1) can be plotted as a function of DG_openº and compared with the plot for a passive helicase (Fig. 3A and SI Fig. 7). We fit the NS3 data to a straight line and obtain the y-intercept. For a passive helicase that has the same y-intercept as that of NS3, the difference between these two plots at the same stepping velocity gives the minimum amounts of free energy reduction brought about by NS3 during active unwinding, which are listed in the third column.

1. Dumont S, Cheng W, Serebrov V, Beran RK, Tinoco I, Jr, Pyle AM, Bustamante C (2006) Nature 439:105-108.

2. Zuker M (2003) Nucleic Acids Res 31:3406-3415.

3. Liphardt J, Onoa B, Smith SB, Tinoco I, Jr, Bustamante C (2001) Science 292:733-737.

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