Center for Structural Biology, National Cancer Institute, Frederick, MD, USA.
RNA Biol. 2022 Jan;19(1):908-915. doi: 10.1080/15476286.2022.2099650.
The RNase III family of dsRNA-specific endonucleases is exemplified by prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. Structures of RNase III (AaRNase III) and Rnt1p (ScRnt1p) show that both enzymes recognize substrates in a sequence-specific manner and propel RNA hydrolysis by two-Mg-ion catalysis. Previously, we created an RNase III variant (EcEEQ) by eliminating the sequence specificity via protein engineering and called it bacterial Dicer for the fact that it produces heterogeneous small interfering RNA cocktails. Here, we present a 1.8-Å crystal structure of a postcleavage complex of EcEEQ, representing a reaction state immediately after the cleavage of scissile bond. The structure not only establishes the structure-and-function relationship of EcEEQ, but also reveals the functional role of a third Mg ion that is involved in RNA hydrolysis by bacterial RNase III. In contrast, the cleavage site assembly of ScRnt1p does not contain a third Mg ion. Instead, it involves two more amino acid side chains conserved among eukaryotic RNase IIIs. We conclude that the EcEEQ structure (this work) represents the cleavage assembly of prokaryotic RNase IIIs and the ScRnt1p structure (PDB: 4OOG), also determined at the postcleavage state, represents the cleavage assembly of eukaryotic RNase IIIs. Together, these two structures provide insights into the reaction trajectory of two-Mg-ion catalysis by prokaryotic and eukaryotic RNase III enzymes.
RNase III 家族的 dsRNA 特异性内切酶以原核 RNase III 和真核 Rnt1p、Drosha 和 Dicer 为代表。RNase III（AaRNase III）和 Rnt1p（ScRnt1p）的结构表明，这两种酶都以序列特异性的方式识别底物，并通过双 Mg 离子催化推动 RNA 水解。以前，我们通过蛋白质工程消除了序列特异性，创建了一种 RNase III 变体（EcEEQ），并因其产生异质小干扰 RNA 鸡尾酒的事实将其称为细菌 Dicer。在这里，我们展示了 EcEEQ 的后切割复合物的 1.8Å 晶体结构，代表了切割后立即的反应状态。该结构不仅建立了 EcEEQ 的结构-功能关系，还揭示了第三个 Mg 离子在细菌 RNase III 中参与 RNA 水解的功能作用。相比之下，ScRnt1p 的切割位点组装不包含第三个 Mg 离子。相反，它涉及到真核 RNase III 中保守的另外两个氨基酸侧链。我们得出结论，EcEEQ 结构（本文工作）代表了原核 RNase III 的切割组装，而 ScRnt1p 结构（PDB：4OOG），也在切割后状态下确定，代表了真核 RNase III 的切割组装。这两个结构共同提供了对原核和真核 RNase III 酶双 Mg 离子催化反应轨迹的深入了解。
