Plots of initial velocity versus substrate concentration: A, hDcr; and B, Δhelicase. C, summary of kinetic values.

Equilibrium filter binding assays for hDcr proteins complexed with 37ab RNA (A) or pre-hlet-7 RNA (B). Values are averages from two independent assays. C, summary of dissociation constants (K_d, nM).

A, Domain structure of hDcr variants; B, 10% SDS-PAGE analysis of recombinant hDcr proteins; C, Single time-point dsRNase activity assays (60 min., 37□, 60 nM protein, 2 nM ³²P-labeled 37ab RNA); M, protein size marker.

A, size exclusion chromatography of the hDcr-TRBP complex formed by incubation of hDcr (molecular weight 219 kDa; 2.5 nmol) and excess TRBP (molecular weight 39 kDa; 9 nmol) in 20 μl dicing buffer for 60 min. on ice; chromatogram peak C, hDcr-TRBP complex; chromatogram peak T, TRBP; chromatogram peak hD, hDcr. SDS-PAGE gel analysis of fractions are shown below each chromatogram. B, multiple-turnover assay for 37ab cleavage using 100 nM dsRNA and 5 nM hDcr or chromatographically pure hDcr-TRBP; the data shown are representative of two independent experiments.

A, Substrates and dicing reactions. Left panels, perfect-duplex dsRNA substrate, 37ab; right panels, hairpin pre-miRNA substrate, pre-hlet-7; asterisks (*) indicate 5′-end labeled with ³²P. hDcr generates two products from RNA 37ab, 22-nt (P₁) or 15-nt (P₂), and one product (P) from pre-hlet-7. Relative rates of P₁ and P₂ production were the same for all hDcrs tested and were combined to give the total cleavage product for 37ab. B, Single-turnover reaction of hDcrs (60 nM) with 2 nM (3000 c.p.m.) duplex RNA 37ab (left panel) or pre-hlet-7 (right panel); values are the average from two independent experiments. Data were fit to the equation S=(a-b)exp(−k_obsdt)+b, where S is the fraction of dsRNA cleaved at each time point, a is the fraction of dsRNA at the beginning of the reaction, b is the fraction of dsRNA at the reaction plateau (t-->∞), and k_obsd is the observed rate constant; k_obsd values (fmol/min) for the 37ab substrate were as follows: hDcr (wildtype), 0.32; hWalker, 0.8; Δhelicase (Δhel), 2; ΔDUF, 0.0034; ΔRBD, 0.2; 2DD, 0.46; k_obsd values (fmol/min) for the pre-hlet-7 substrate were as follows: hDcr (wildtype), 3; hWalker, 2; Δhelicase, 2.2; ΔDUF, 2.6; ΔRBD, 0.74; 2DD, 0.74; C, summary of initial reaction rates of hDcr variants calculated for 20% substrate cleavage; S, RNA substrate; FL, wild-type hDicer; hW, hWalker mutant; dhel, Δhelicase mutant; dDUF, ΔDUF mutant; dRBD, ΔRBD mutant.