Autoinhibition of human dicer by its internal helicase domain
Autoinhibition of human dicer by its internal helicase domain
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DOI：
10.1016/j.jmb.2008.05.005
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发表时间：
2008-06-27
影响因子：
5.6
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
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作者：
Ma, Enbo;MacRae, Ian J.;Doudna, Jennifer A.
Dicer是核糖核酸酶III家族的一员，它将双链RNA底物加工成类似于21-至27-nt的产物，通过RNA干扰触发序列导向基因沉默。虽然Dicer的RNA识别和长度特异性切割机制已经建立，但其切割活性的调节方式尚不清楚。在这里，我们发现人类Dicer的n端结构域，与DExD/H-box解旋酶同源，大大降低了底物切割的速度。该结构域的缺失或突变在单次和多次翻转试验中激活人类Dicer。缺失结构的催化效率(k（cat)/ k(m)）比完整酶提高了65倍。动力学分析表明，这种活化几乎完全是由于k（cat）的增强。在与DExD/H-box结构域物理相互作用的TAR-RNA结合蛋白存在的情况下，观察到全长Dicer酶对催化的适度刺激。这些结果表明，DExD/H-box结构域可能会破坏Dicer活性位点的功能，直到结构重排发生，可能是在与其分子伙伴组装时发生的。(C) 2008 Elsevier Ltd版权所有。
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.