Autoinhibition of Human Dicer by Its Internal Helicase Domain

• Published in: Journal of molecular biology 2008-06, Vol.380 (1), p.237-243
• Main Authors: Ma, Enbo, MacRae, Ian J., Kirsch, Jack F., Doudna, Jennifer A.
• Format: Article
• Language: English
• Subjects: Catalysis; DEAD-box RNA Helicases - antagonists & inhibitors; DEAD-box RNA Helicases - chemistry; dicer; Endoribonucleases - antagonists & inhibitors; Endoribonucleases - chemistry; helicase; Humans; Kinetics; Protein Binding; Protein Structure, Tertiary; ribonuclease; Ribonuclease III; RNA, Double-Stranded - metabolism; RNA-Binding Proteins - metabolism; RNAi; Sequence Deletion; Substrate Specificity
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.05.005

Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into ∼21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (kcat/Km) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in kcat. Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.