Figure 5.

Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. (A) ATP and Mg ions are required for both DCL3 and DCL4 activities. Dicer assays for 50 nt dsRNA with a 1 nt 3′ overhang as a substrate were performed in reaction mixtures under various conditions. The standard Dicer buffer contained 30 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM MgCl₂, 5 mM ATP and 1 mM GTP. To this buffer, 5 mM ATPγS or 4 mM EDTA was added. Sodium chloride (B) and inorganic phosphate (C) enhance DCL3 activity but inhibit DCL4 activity. Dicer assays were performed with various concentrations of NaCl (B) or KH₂PO₄ (C). Cleavage products from 50 nt dsRNA with a 2 nt 3′ overhang were analysed. The 21 nt RNA producing activity (DCL4 activity) was calculated as the relative band intensity of 21 nt RNA with respect to the intensity of all bands in each lane, and the 24 nt RNA producing activity (DCL3 activity) was calculated using the same equation. Values are means ± standard deviation for three independent experiments.