Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.
J Biol Chem. 2010 Aug 13;285(33):25363-71. doi: 10.1074/jbc.M110.117010. Epub 2010 Jun 7.
RNA helicases are proteins essential to almost every facet of RNA metabolism, including the gene-silencing pathways that employ small RNAs. A phylogenetically related group of helicases is required for the RNA-silencing mechanism in Caenorhabditis elegans. Dicer-related helicase 3 (DRH-3) is a Dicer-RIG-I family protein that is essential for RNA silencing and germline development in nematodes. Here we performed a biochemical characterization of the ligand binding and catalytic activities of DRH-3 in vitro. We identify signature motifs specific to this family of RNA helicases. We find that DRH-3 binds both single-stranded and double-stranded RNAs with high affinity. However, the ATPase activity of DRH-3 is stimulated only by double-stranded RNA. DRH-3 is a robust RNA-stimulated ATPase with a k(cat) value of 500/min when stimulated with short RNA duplexes. The DRH-3 ATPase may have allosteric regulation in cis that is controlled by the stoichiometry of double-stranded RNA to enzyme. We observe that the DRH-3 ATPase is stimulated only by duplexes containing RNA, suggesting a role for DRH-3 during or after transcription. Our findings provide clues to the role of DRH-3 during the RNA interference response in vivo.
RNA 解旋酶是几乎所有 RNA 代谢过程中必不可少的蛋白质，包括利用小 RNA 的基因沉默途径。在秀丽隐杆线虫中，一组系统发生上相关的解旋酶对于 RNA 沉默机制是必需的。Dicer 相关解旋酶 3（DRH-3）是一种 Dicer-RIG-I 家族蛋白，对于线虫中的 RNA 沉默和生殖细胞发育是必需的。在这里，我们在体外对 DRH-3 的配体结合和催化活性进行了生化表征。我们确定了这个 RNA 解旋酶家族特有的特征基序。我们发现 DRH-3 可以高亲和力结合单链和双链 RNA。然而，只有双链 RNA 才能刺激 DRH-3 的 ATP 酶活性。DRH-3 是一种强大的 RNA 刺激的 ATP 酶，当用短的 RNA 双链体刺激时，k(cat) 值为 500/min。DRH-3 ATP 酶可能在顺式中具有变构调节，由双链 RNA 与酶的比例控制。我们观察到只有含有 RNA 的双链体才能刺激 DRH-3 的 ATP 酶，这表明 DRH-3 在转录过程中或之后发挥作用。我们的发现为 DRH-3 在体内 RNA 干扰反应中的作用提供了线索。
