P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).
P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).
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DOI：
10.1016/j.jmb.2015.12.018
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发表时间：
2016-01-29
影响因子：
5.6
通讯作者：
De La Cruz EM
中科院分区：
文献类型：
--
作者：
Wong EV;Cao W;Vörös J;Merchant M;Modis Y;Hackney DD;Montpetit B;De La Cruz EM
mRNA从细胞核的输出依赖于DEAD盒蛋白Dbp 5/DDX 19的ATP酶活性。虽然Dbp 5单独具有可测量的ATP酶活性，但几种调节因子（例如，RNA、核孔蛋白和内源性小分子InsP 6）在体外和体内调节催化活性以促进mRNA输出。内在的和调节激活Dbp 5 ATP酶循环的分析是必要的，以确定这些因素如何控制Dbp 5和mRNA的出口。在这里，我们报告的动力学和平衡分析酿酒酵母Dbp 5 ATP酶循环，包括Dbp 5活性的RNA的影响。这些数据表明，ATP在快速平衡中以与稳态循环的KM相当的结合亲和力（KD ~ 4 mM）弱结合Dbp 5，而ADP结合更紧密一个数量级（KD ~ 0.4mM）。整体固有稳态循环速率常数（kcat）受到缓慢、近乎不可逆的ATP水解和甚至更慢的后续磷酸盐释放的限制。RNA增加kcat和限速Pi释放20倍，尽管Pi释放继续限制RNA存在下的稳态循环，结合RNA结合。总之，这项工作确定RNA结合和Pi释放的Dbp 5 ATP酶循环内的重要的生化转换，并提供了一个框架，调查Dbp 5和mRNA的出口是由调节因子调制的手段。mRNA从细胞核输出需要DEAD盒蛋白Dbp 5/DDX 19 ATP酶活性。内源性Dbp 5 ATP酶的动力学和热力学揭示了RNA对Dbp 5的影响。内源性Dbp 5 ATP酶受缓慢的ATP水解和较慢的Pi释放的限制。RNA激活Pi的释放，但它和RNA结合仍然限制RNA刺激的ATP酶。RNA结合和Pi释放定义了RNA刺激的Dbp 5 ATP酶，用于进一步调节。
mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT ~ 4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD ~ 0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors. mRNA export from the nucleus requires DEAD-box protein Dbp5/DDX19 ATPase activity. Kinetics and thermodynamics of intrinsic Dbp5 ATPase reveal RNA's effect on Dbp5. Intrinsic Dbp5 ATPase is limited by slow ATP hydrolysis and slower Pi release. RNA activates Pi release, but it and RNA binding still limit RNA-stimulated ATPase. RNA binding and Pi release define RNA-stimulated Dbp5 ATPase for further regulation.