Structural Mechanism of Substrate RNA Recruitment in H/ACA RNA-Guided Pseudouridine Synthase
Structural Mechanism of Substrate RNA Recruitment in H/ACA RNA-Guided Pseudouridine Synthase
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DOI：
10.1016/j.molcel.2009.05.005
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发表时间：
2009-05-29
期刊：
影响因子：
16
通讯作者：
Ye, Keqiong
中科院分区：
文献类型：
--
作者：
Duan, Jingqi;Li, Ling;Ye, Keqiong
H/ACA RNA与Cbf5、Nop10、L7Ae和Gar1蛋白形成核糖核蛋白复合体(RNP)，并在细胞RNA中引导尿苷定点转化为假尿苷。H/ACA RNP的晶体结构表明，底物是通过与引导RNA和必需蛋白质接触的序列特异性配对而被招募的。底物结合导致预设的假尿路口袋的重组，以及H/ACA RNA的PUA结构域和下茎的适应性移动。此外，拇指环从无底物RNP结构中的Gar1结合状态翻转，以与底物紧密结合。突变和酶动力学分析表明，Gar1和拇指在底物周转，特别是在产物释放中起着关键作用。与tRNA PSI55合成酶TRUB的比较揭示了RNA引导的假尿苷合成酶和独立的假尿苷合成酶之间的结构保守和适应，并为Cbf5的导向非依赖活性提供了洞察力。
H/ACA RNAs form ribonucleoprotein complex (RNP) with proteins Cbf5, Nop10, L7Ae, and Gar1 and guide site-specific conversion of uridine into pseudouridine in cellular RNAs. The crystal structures of H/ACA RNP with substrate bound at the active site cleft reveal that the substrate is recruited through sequence-specific pairing with guide RNA and essential protein contacts. Substrate binding leads to a reorganization of a preset pseudouridylation pocket and an adaptive movement of the PUA domain and the lower stem of the H/ACA RNA. Moreover, a thumb loop flips from the Gar1-bound state in the substrate-free RNP structure to tightly associate with the substrate. Mutagenesis and enzyme kinetics analysis suggest a critical role of Gar1 and the thumb in substrate turnover, particularly in product release. Comparison with tRNA Psi 55 synthase TruB reveals the structural conservation and adaptation between an RNA-guided and stand-alone pseudouridine synthase and provides insight into the guide-independent activity of Cbf5.