FIG 6.
Utp14 activates Dhr1 unwinding activity in vitro. (A) Cartoon of U3-ETS2 substrate used for unwinding assays. Full-length U3 snoRNA was used for the reactions in panels B and C. (B) Representative unwinding reactions stopped after 20 min. EMSAs separated the 32P-labeled ETS2 free (unwound) from its duplex form. The RT reaction mixture contained 50 nM Dhr1 (WT or mutant), 200 nM Utp14 (WT or mutant), 1 mM ATP, and ≤0.3 nM U3-ETS2 duplex with other reagents described in Materials and Methods. (C) Fraction unwound was plotted as a function of time after addition of ATP and either Utp14, Dhr1, or Dhr1 in the presence of either Utp14, Utp14Multi-Ala, or Utp14Δ719-780 or after addition of Dhr1 and Utp14 in the absence of ATP. (D) Fraction unwound was plotted as a function of time after addition of ATP and Dhr1D516A/E517A in the absence or presence of Utp14.