Autoinhibition of human dicer by its internal helicase domain

被引:171
作者
Ma, Enbo [1 ]
MacRae, Ian J. [1 ,2 ]
Kirsch, Jack F. [1 ,3 ]
Doudna, Jennifer A. [1 ,2 ,3 ]
机构
[1] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA
[2] Univ Calif Berkeley, Howard Hughes Med Inst, Berkeley, CA 94720 USA
[3] Univ Calif Berkeley, Dept Chem, Berkeley, CA 94720 USA
关键词
ribonuclease; dicer; RNAi; helicase;
D O I
10.1016/j.jmb.2008.05.005
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into similar to 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. (C) 2008 Elsevier Ltd. All rights reserved.
引用
收藏
页码:237 / 243
页数:7
相关论文
共 19 条
[1]   Role for a bidentate ribonuclease in the initiation step of RNA interference [J].
Bernstein, E ;
Caudy, AA ;
Hammond, SM ;
Hannon, GJ .
NATURE, 2001, 409 (6818) :363-366
[2]   RNase III enzymes and the initiation of gene silencing [J].
Carmell, MA ;
Hannon, GJ .
NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2004, 11 (03) :214-218
[3]   DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold [J].
Dlakic, Mensur .
BIOINFORMATICS, 2006, 22 (22) :2711-2714
[4]   Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA [J].
FerreDAmare, AR ;
Doudna, JA .
NUCLEIC ACIDS RESEARCH, 1996, 24 (05) :977-978
[5]   TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing [J].
Haase, AD ;
Jaskiewicz, L ;
Zhang, HD ;
Lainé, S ;
Sack, R ;
Gatignol, A ;
Filipowicz, W .
EMBO REPORTS, 2005, 6 (10) :961-967
[6]   Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C-elegans [J].
Ketting, RF ;
Fischer, SEJ ;
Bernstein, E ;
Sijen, T ;
Hannon, GJ ;
Plasterk, RHA .
GENES & DEVELOPMENT, 2001, 15 (20) :2654-2659
[7]   The role of PACT in the RNA silencing pathway [J].
Lee, Y ;
Hur, I ;
Park, SY ;
Kim, YK ;
Suh, MR ;
Kim, VN .
EMBO JOURNAL, 2006, 25 (03) :522-532
[8]   R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway [J].
Liu, QH ;
Rand, TA ;
Kalidas, S ;
Du, FH ;
Kim, HE ;
Smith, DP ;
Wang, XD .
SCIENCE, 2003, 301 (5641) :1921-1925
[9]   Ribonuclease revisited: structural insights into ribonuclease III family enzymes [J].
MacRae, Ian J. ;
Doudna, Jennifer A. .
CURRENT OPINION IN STRUCTURAL BIOLOGY, 2007, 17 (01) :138-145
[10]   Structural basis for double-stranded RNA processing by dicer [J].
MacRae, IJ ;
Zhou, KH ;
Li, F ;
Repic, A ;
Brooks, AN ;
Cande, WZ ;
Adams, PD ;
Doudna, JA .
SCIENCE, 2006, 311 (5758) :195-198