Characterization of the mammalian DEAD-box protein DDX5 reveals functional conservation with S. cerevisiae ortholog Dbp2 in transcriptional control and glucose metabolism

The CTE confers higher duplex unwinding activity to human DDX5 over S. cerevisiae Dbp2. (A) MBP-DDX5-GST, DDX5ΔCTE, and Dbp2 have identical functional K_m’s for ATP in unwinding assays. RNA unwinding assays were conducted using 300 nM recombinant, purified proteins and the indicated amount of ATP/MgCl₂ in the presence of 30 mM NaCl at 19°C. The curves for observed unwinding rates versus ATP were fitted to a binding isotherm: Y = K_obs(max) × X/K_m^(unw) + X. (B) DDX5 shows higher unwinding activity than DDX5ΔCTE or Dbp2 in “low-salt” conditions. (1) Helicase unwinding assays were conducted at 19°C in the presence of 30 mM NaCl with indicated concentrations of purified recombinant MBP-DDX5-GST, DDX5ΔCTE, Dbp2, or MBP-DDX5ΔCTE-GST. (2) Table of maximum observed unwinding rates and the functional dissociation constants. First, the observed rates (k_obs^(Unw)) were determined by plotting the fraction of single-stranded (ss) RNA against reaction time and fitting the unwinding curves to the following equation: Y = Y_max × [1 − exp(−k_obs^(Unw) × X)]. To calculate the maximum observed rates (K_obs(max)^(Unw)) and functional dissociation constants (K_d^(Unw)), we plotted the observed rates as a function of the protein concentration and fitted the data to a sigmoidal binding isotherm [Y = K_obs(max)^(Unw) × X^h/(K_d^(Unw)h + X^h)] to generate functional binding curves. The Hill coefficients for these enzymes range from 1.5 to 1.8. (C) DDX5 is an active helicase in “near-physiological salt” conditions. Unwinding assays using 500 nM purified recombinant MBP-DDX5-GST, DDX5ΔCTE, or Dbp2 were performed in the presence of 100 mM NaCl at 30°C. The observed rates were determined as above. n.d., means not determined, when unwinding curves were not convergent. Data above show the mean ± standard deviation (SD) of 2–3 independent replicates.