FIGURE 7.

Mtr4p but not Mtr4-20p and Mtr4-21p unwinds RNA duplexes with a 3′-to-5′ polarity in an ATP-dependent fashion. (A) Representative PAGE of an unwinding reaction of a 16-bp duplex [S16-3′] with a 3′-single-stranded overhang by wild-type Mtr4p. Cartoons on the left represent the mobility of duplex and the single-stranded radiolabeled RNA; the asterisk indicates the radiolabel. Reactions were assembled with Mtr4p and without ATP, and an aliquot removed at time zero. After ATP addition, aliquots were removed at the times shown. Complete dissociation of the duplex is shown after incubating the RNA briefly at 95°C. (B) Time courses of unwinding reactions by wild-type Mtr4p for 16-bp duplexes with 3′- and 5′-single-stranded overhangs with and without ATP (inset). Sequences of duplexes and single stranded overhangs were identical (Materials and Methods). Significant unwinding was seen only for the substrate with the 3′-overhang (•). The solid line through these points indicates the best fit to a first-order reaction (Yang and Jankowsky 2005), yielding the reaction amplitude A = 0.65 ± 0.02, and the observed unwinding rate constant k′_unw = 0.057 ± 0.003 min⁻¹. (C) Representative PAGE of unwinding reactions with a 19-bp substrate with a 3′-overhang [S19-3′] with wild-type Mtr4p and Mtr4-20p and Mtr4-21p. For the mutant proteins, only aliquots removed after 60 min are shown. Wild-type Mtr4p unwinds the 19-bp substrate with an observed rate constant (k′_unw = 0.054 ± 0.007 min⁻¹) highly similar to that seen with the 16-bp duplex, but the reaction amplitude is lower (A = 0.39 ± 0.03) than for the 16-bp duplex.