Autoinhibition of human dicer by its internal helicase domain.
Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 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 (k(cat)/K(m)) 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 k(cat). 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.
Ma
Enbo
E
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3206, USA.
MacRae
Ian J
IJ
Kirsch
Jack F
JF
Doudna
Jennifer A
JA
D002384:Catalysis; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D043244:Ribonuclease III; D017384:Sequence Deletion; D013379:Substrate Specificity; D000099253:Trans-Activation Responsive RNA-Binding Protein
D053487:DEAD-box RNA Helicases; D004722:Endoribonucleases; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein; C408087:DICER1 protein, human