Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation.
Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation.
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Box H/ACA RNA引导的假尿苷形成反应途径的动力学和热力学表征
DOI：
10.1093/nar/gks882
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发表时间：
2012-11
影响因子：
14.9
通讯作者：
Zhao XS
中科院分区：
文献类型：
--
作者：
Yang X;Duan J;Li S;Wang P;Ma S;Ye K;Zhao XS
盒H/ACA RNA引导的假尿苷合酶是一种复杂的核糖核蛋白酶，其通过引导RNA和催化亚基Cbf 5两者募集底物。结构研究揭示了多种构象的酶，但反应途径的定量描述仍然缺乏。使用荧光相关光谱，我们在这里测量的平衡解离常数和动力学协会和解离速率的底物和产物复合物模仿各种反应中间状态。这些数据支持由Cbf 5的拇指环调节的底物加载和产物释放的顺序模型。尿苷底物首先主要通过与向导RNA相互作用结合，然后加载到活性位点，同时逐渐与拇指相互作用。修饰后，在靶位点从尿苷到假尿苷的微妙化学结构变化触发拇指的释放，导致主要由指导RNA结合的产物的中间复合物。通过剖析Gar 1在底物周转的各个步骤中的作用，我们发现Gar 1在催化中起着重要作用，并且还加速了产物释放约2倍。我们的生物物理结果与以前的结构知识整合到一个连贯的反应途径H/ACA RNA引导的pseudouridylation。
The box H/ACA RNA-guided pseudouridine synthase is a complicated ribonucleoprotein enzyme that recruits substrate via both the guide RNA and the catalytic subunit Cbf5. Structural studies have revealed multiple conformations of the enzyme, but a quantitative description of the reaction pathway is still lacking. Using fluorescence correlation spectroscopy, we here measured the equilibrium dissociation constants and kinetic association and dissociation rates of substrate and product complexes mimicking various reaction intermediate states. These data support a sequential model for substrate loading and product release regulated by the thumb loop of Cbf5. The uridine substrate is first bound primarily through interaction with the guide RNA and then loaded into the active site while progressively interacted with the thumb. After modification, the subtle chemical structure change from uridine to pseudouridine at the target site triggers the release of the thumb, resulting in an intermediate complex with the product bound mainly by the guide RNA. By dissecting the role of Gar1 in individual steps of substrate turnover, we show that Gar1 plays a major role in catalysis and also accelerates product release about 2-fold. Our biophysical results integrate with previous structural knowledge into a coherent reaction pathway of H/ACA RNA-guided pseudouridylation.
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影响因子：
4.5
作者：
Wright, Jaden R.;Keffer-Wilkes, Laura C.;Kothe, Ute
通讯作者：
Kothe, Ute
影响因子：
10.5
作者：
Baker, DL;Youssef, OA;Terns, MP
通讯作者：
Terns, MP
影响因子：
2.9
作者：
Huang, LX;Pookanjanatavip, M;Santi, DV
通讯作者：
Santi, DV
直接测量悬垂寡核苷酸分子内碰撞正向和反向扩散的速率和势垒
影响因子：
3.3
作者：
Qu, Peng;Yang, Xinxing;Zhao, Xin Sheng
通讯作者：
Zhao, Xin Sheng
DOI：
10.1073/pnas.0701534104
发表时间：
2007-04-17
影响因子：
11.1
作者：
Wu, Haihong;Feigon, Juli
通讯作者：
Feigon, Juli