Song He, Fang Xianyang, Jin Lan, Shaw Gary X, Wang Yun-Xing, Ji Xinhua
Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Structure. 2017 Feb 7;25(2):353-363. doi: 10.1016/j.str.2016.12.013. Epub 2017 Jan 19.
Double-stranded RNA (dsRNA)-specific RNase III proteins are required for RNA maturation and gene regulation. The mechanism of prokaryotic RNase IIIs has been well characterized, but how eukaryotic RNase IIIs (exemplified by Rnt1p, Drosha, and Dicer) work is less clear. Recently, we reported the crystal structure of Rnt1p in complex with RNA, revealing a double-ruler mechanism for substrate selection. Here, we present more structures of Rnt1p, either RNA free or RNA bound, featuring two major conformations of the enzyme. Using these structures with existing data, we describe the functional cycle of Rnt1p in five steps, selecting, loading, locking, cleavage, and releasing. We also describe atomic details of the two-Mg-ion catalytic mechanism that is applicable to all eukaryotic RNase III enzymes. Overall, our results indicate that substrate selection is achieved independent of cleavage, allowing the recognition of substrates with different structures while preserving the basic mechanism of cleavage.
双链RNA（dsRNA）特异性核糖核酸酶III蛋白对于RNA成熟和基因调控是必需的。原核核糖核酸酶III的机制已得到充分表征，但真核核糖核酸酶III（以Rnt1p、Drosha和Dicer为例）的工作方式尚不清楚。最近，我们报道了Rnt1p与RNA复合物的晶体结构，揭示了一种底物选择的双尺机制。在这里，我们展示了更多Rnt1p的结构，包括游离RNA或结合RNA的结构，其呈现出该酶的两种主要构象。利用这些结构和现有数据，我们分五步描述了Rnt1p的功能循环，即选择、加载、锁定、切割和释放。我们还描述了适用于所有真核核糖核酸酶III的双镁离子催化机制的原子细节。总体而言，我们的结果表明底物选择独立于切割实现，允许识别不同结构的底物，同时保留切割的基本机制。
