Abstract：
The highly conserved Superfamily 1 (SF1) and Superfamily 2 (SF2) nucleic acid-dependent ATPases, are ubiquitous motor proteins with central roles in DNA and RNA metabolism (Jankowsky & Fairman, 2007). These enzymes require RNA or DNA binding to stimulate ATPase activity, and the conformational changes that result from this coupled behavior are linked to a multitude of processes that range from nucleic acid unwinding to the flipping of macromolecular switches (Pyle, 2008, 2011). Knowledge about the relative affinity of nucleic acid ligands is crucial for deducing mechanism and understanding biological function of these enzymes. Because enzymatic ATPase activity is directly coupled to RNA binding in these proteins, one can utilize their ATPase activity as a simple reporter system for monitoring functional binding of RNA or DNA to an SF1 or SF2 enzyme. In this way, one can rapidly assess the relative impact of mutations in the protein or the nucleic acid and obtain parameters that are useful for setting up more quantitative direct binding assays. Here, we describe a routine method for employing NADH-coupled enzymatic ATPase activity to obtain kinetic parameters reflecting apparent ATP and RNA binding to an SF2 helicase. First, we provide a protocol for calibrating an NADH-couple ATPase assay using the well-characterized ATPase enzyme hexokinase, which a simple ATPase enzyme that is not coupled with nucleic acid binding. We then provide a protocol for obtaining kinetic parameters (KmATP, Vmax and KmRNA) for an RNA-coupled ATPase enzyme, using the double-stranded RNA binding protein RIG-I as a case-study. These approaches are designed to provide investigators with a simple, rapid method for monitoring apparent RNA association with SF2 or SF1 helicases.
摘要：
高度保守的超家族1（SF1）和超家族2（SF2）核酸依赖性ATP酶，是普遍存在的运动蛋白，在DNA和RNA代谢中具有核心作用(Jankowsky&Fairman，2007).这些酶需要RNA或DNA结合来刺激ATP酶活性，这种耦合行为导致的构象变化与许多过程有关，这些过程从核酸展开到大分子开关的翻转(派尔，2008、2011)。关于核酸配体的相对亲和力的知识对于推断这些酶的机制和理解这些酶的生物学功能至关重要。因为酶促ATP酶活性直接与这些蛋白质中的RNA结合偶联，人们可以利用它们的ATP酶活性作为用于监测RNA或DNA与SF1或SF2酶的功能性结合的简单报告系统。这样,可以快速评估蛋白质或核酸中突变的相对影响，并获得可用于建立更定量的直接结合测定的参数。这里,我们描述了使用NADH偶联的酶促ATPase活性来获得反映表观ATP和RNA与SF2解旋酶结合的动力学参数的常规方法。首先,我们提供了使用充分表征的ATP酶己糖激酶校准NADH偶联ATP酶测定的方案，一种简单的ATP酶，不与核酸结合。然后，我们提供了一个获得动力学参数的协议(KmATP，Vmax和KmRNA）用于RNA偶联的ATP酶，使用双链RNA结合蛋白RIG-I作为案例研究。这些方法旨在为调查人员提供一种简单的，用于监测与SF2或SF1解旋酶的表观RNA关联的快速方法。
