Motif Ib is not required for RNA binding or ATPase activity but is required for unwinding a 3′ overhang duplex. (A) Mutations in the tip of the 3′HP encompassing motif Ib (R177A or R177A/F178A) compromise unwinding of a 21-bp RNA/DNA duplex having a 27-nt 3′ RNA overhang. (B) Mutations in motif Ib permit RNA binding. Binding of Prp43 to the RNA unwinding substrate used in panel A was assayed by EMSA under conditions similar to the unwinding reactions but in the absence of ATP. The Prp43p–RNA complex is indicated. (C) Mutations in motif Ib permit ATPase activity. RNA-stimulated, Prp43-dependent ATPase activity was analyzed by TLC. The mutation E216A of motif II, the Walker B motif, was used as a negative control. (D) Unwinding of either a 3′ or 5′ unstructured 32-nt RNA overhang substrate with an identical 28-mer duplex. Heat denaturation of the duplex (Δ) was used as a positive control for unwinding. A line separating lanes 7 and 8 or lanes 15 and 16 indicates that a single gel image was cut and arranged to juxtapose relevant lanes.
