FIGURE 6.
Top, the general scheme of formation of functional NS3-RNA complex, where S is an RNA substrate; E is NS3 protein; P is unwound product; EnS* is the functional complex, and n is number of NS3 molecules bound to the RNA substrate in the functional complex. Bottom, the experimental design of functional complex formation experiments. The RNA substrate is preincubated with NS3 for variable time Δt, after which unwinding is initiated by the addition of ATP and trap oligonucleotide. The trap oligonucleotide is included to sequester any unbound protein and to prevent re-binding of NS3 to the RNA substrate during the unwinding reaction (single cycle unwinding conditions). The unwinding reactions are allowed to proceed to completion (90 s), after which unwinding amplitudes (Aunw) are determined for each Δt by resolving the unwound products by native PAGE.