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首页> 外文期刊>Journal of Molecular Biology >The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA
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The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

机译：DEAD-box rRNA解旋酶DbpA的ATPase循环机制

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摘要

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a gamma-phosphate attack mechanism. ADP-Pi and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-Pi to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by Pi release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-Pi-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases. (c) 2007 Elsevier Ltd. All rights reserved.
机译：DEAD-box蛋白是使双链RNA不稳定和解旋的ATPase酶。 ATPase循环参数的定量知识对于开发解旋酶活性模型至关重要。但是，有关定义RNA解旋酶ATP酶循环速率和平衡常数的信息有限，包括人口稠密的生化中间体的分布，限制酶促反应循环的催化步骤以及如何利用ATP和RNA相互作用。我们提出了定量的动力学和平衡特征的DbpA，牵连核糖体生物发生的DEAD框rRNA解旋酶的核糖体RNA（rRNA）激活ATPase循环机制。 rRNA通过促进与水解相关的ATP结合后的构象变化，激活DbpA的ATPase活性。结合的ATP的化学裂解是可逆的，并通过γ-磷酸盐攻击机制发生。 ADP-Pi和RNA的结合表现出很强的热力学耦合性，这导致DbpA-ADP-Pi结合的rRNA的亲和力比结合的ATP，ADP或不存在核苷酸的亲和力高10倍以上。 DbpA的rRNA激活的稳态ATPase循环受ATP水解和Pi释放的限制，而Pi的释放速率相当。因此，稳态循环过程中主要是填充的生化状态是与ATP和ADP-Pi结合的中间体。 ATPase循环转变的热力学连锁分析有利于建立模型，其中rRNA双链体不稳定与强rRNA和核苷酸结合有关。提出的DbpA ATPase循环反应机理的分析为建立有关DEAD-box解旋酶功能和分子机理的可检验假设提供了严格的动力学和热力学基础。 （c）2007 Elsevier Ltd.保留所有权利。

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