Kinetic analysis of CDK9 16-nt RISC. (A) CDK9 RISC cleavage of target mRNA depends on substrate concentration. Efficiency of target mRNA cleavage by CDK9 RISC programmed by 16-nt siRNA at various substrate concentrations. (B) CDK9 RISC cleavage of target mRNA shows Michaelis–Menten kinetics. Michaelis–Menten analysis of 16-nt RISC. The initial velocity of cleavage product formation was determined from the slope of a curve fit to the cleavage reactions at 0–30 min as shown in (A). (C) The 16-nt siRNA programs more RISC than 19-nt siRNA or 29-nt shRNA. Concentrations of active CDK9 RISC programmed by 16-nt RNA, 19-nt siRNA, or 29-nt shRNA were determined by 2′-O-Me inhibition in vitro cleavage assay. (D) Kinetic analysis of 16-nt CDK9 RISC. K m and V max were determined by Michaelis–Menten analysis as shown in (B).
