Ma Enbo, MacRae Ian J, Kirsch Jack F, Doudna Jennifer A
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3206, USA.
J Mol Biol. 2008 Jun 27;380(1):237-43. doi: 10.1016/j.jmb.2008.05.005. Epub 2008 May 8.
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.
Dicer是核糖核酸酶III家族的一种酶，它将双链RNA底物加工成约21至27个核苷酸的产物，这些产物通过RNA干扰引发序列定向的基因沉默。尽管已经确定了Dicer识别RNA和进行长度特异性切割的机制，但Dicer切割活性的调节方式尚不清楚。在这里，我们表明，人Dicer的N端结构域与DExD/H-box解旋酶同源，它会显著降低底物切割的速率。在单轮和多轮实验中，该结构域的缺失或突变都会激活人Dicer。缺失构建体的催化效率（k(cat)/K(m)）比完整酶提高了65倍。动力学分析表明，这种激活几乎完全是由于k(cat)的增强。在存在与DExD/H-box结构域发生物理相互作用的TAR-RNA结合蛋白的情况下，观察到全长Dicer酶对催化有适度的刺激作用。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，直到发生结构重排，这可能是在与分子伴侣组装时发生的。
