DEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N- and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions.

中文翻译：

---

HIV-1 gRNA 是一种生物底物，揭示了 DDX3X 生化活性的效力。


DEAD-box解旋酶通过协助RNA和核糖核蛋白的合成、折叠、功能甚至降解或分解，在许多RNA和核糖核蛋白的代谢中发挥核心作用。它们与各种现象有关，并且通常很难从体内研究中合理解释它们的分子作用。一旦在体外纯化，它们中的大多数仅表现出边际活性和较差的特异性。目前的模型是，它们通过其本质上无序的结构域与特定 RNA 或蛋白质的相互作用来获得特异性和活性。 DDX3 是一种 DEAD-box 细胞解旋酶，参与 HIV 病毒周期的多个步骤，包括转录、RNA 输出到细胞质和翻译。在这项研究中，我们研究了生物底物背景下的 DDX3 生化特性。 DDX3 被过表达、纯化，并使用模型底物和生物底物 HIV-1 gRNA 来表征其酶活性及其 RNA 结合特性。 DDX3 在生物底物背景下的生化表征将 HIV-1 gRNA 识别为特定底物的罕见例子，并揭示了 DDX3 ATP 酶活性的程度。 DDX3 结合能力的分析表明其结合能力与其生化活性之间存在意外的分离。我们进一步证明，DDX3 与 HIV-1 gRNA 的相互作用既依赖于特定的 RNA 决定簇，也依赖于蛋白质的无序 N 端和 C 端区域。这些发现为 DDX3 生化活性支持其多种细胞功能的潜力提供了新的线索。