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首页> 外文期刊>Journal of Molecular Biology >P-i Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5)
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P-i Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5)

机译：P-i释放限制了DEAD-Box蛋白5（Dbp5）的内在和RNA刺激的ATPase循环。

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摘要

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 InsP(6)) 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 (K-T similar to 4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (K-D similar to 0.4 mM). The overall intrinsic steady-state cycling rate constant (k(cat)) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting P-i release 20-fold, although P-i release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and P-i 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. (C) 2016 Elsevier Ltd. All rights reserved.
机译：从细胞核中输出的mRNA取决于DEAD-box蛋白Dbp5 / DDX19的ATPase活性。尽管Dbp5单独具有可测量的ATPase活性，但一些调节因子（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的输出。必须对内在的和调节子激活的Dbp5 ATPase循环进行分析，以定义这些因素如何控制Dbp5和mRNA的输出。在这里，我们报告了酿酒酵母Dbp5 ATPase周期的动力学和平衡分析，包括RNA对Dbp5活性的影响。这些数据表明，ATP在快速平衡中弱结合Dbp5，其结合亲和力（K-T类似于4 mM）与稳态循环中的KM相当，而ADP则更紧密地结合了一个数量级（K-D类似于0.4 mM）。整体的固有稳态循环速率常数（k（cat））受缓慢的，几乎不可逆的ATP水解甚至是随后的磷酸盐释放速度的限制。 RNA使kcat和限速P-1释放增加20倍，尽管P-1释放继续限制存在RNA时的稳态循环以及RNA结合。总之，这项工作将RNA结合和P-i释放确定为Dbp5 ATPase循环内的重要生化转变，并提供了一个框架，用于研究调控因子调节Dbp5和mRNA输出的方法。 （C）2016 Elsevier Ltd.保留所有权利。

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