Figure 4. Binding and Cleavage of a dsRNA Duplex.
(A) Structural alignment of apo-Dcr1ΔC (green) and AaRNase III–dsRNA complex (tan) based on their RNase III domains.
(B) Close-up view of the VL-1 and VL-2 loops. The dsRNA is represented with a sphere model. The numbering of secondary structure elements is as in Figure 3A.
(C) Dicing activity of variable-loop mutants under standard single-turnover conditions. Body-labeled 500 bp dsRNA was incubated for 5 s or 30 min with buffer only (−), wild-type Dcr1ΔC (WT), or Dcr1ΔC variants with the indicated GiDicer loop substitutions.
(D) Cleavage reactions following body- or end-labeled 70 bp dsRNA substrates. Substrates were incubated with buffer only (−), K. polysporus Dcr1ΔC (Kp; standard single-turnover conditions), or H. sapiens Dicer (Hs). HsDicer generates ~21–22 nt siRNA products and ~40–50 nt intermediates as indicated; a ~25 nt product of unknown origin is also observed (*).
(E) A pair of Dcr1ΔC dimers (green and yellow) modeled with dsRNA (sphere model). The anticipated 23 nt siRNA product is highlighted (cyan). The dsRBD is not shown for clarity.
(F) Protein cross-linking analyses of Dcr1ΔC oligomerization. E224Q Dcr1ΔC was incubated with buffer only (−) or the indicated nucleic acid before crosslinking with the indicated glutaraldehyde (GA) concentration. Reactions were analyzed by SDS-PAGE and visualized by silver staining. Also shown is the migration of protein standards with the indicated molecular weights.
See also Figure S4.