Laboratoire de Physique de l'Ecole normale supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université Paris-Diderot, Sorbonne Paris Cité, Paris, France.
IBENS, Département de biologie, École normale supérieure, CNRS, INSERM, PSL Research University, 75005 Paris, France.
Nucleic Acids Res. 2019 Apr 23;47(7):3699-3710. doi: 10.1093/nar/gkz057.
DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.
DEAD-box 解旋酶参与 RNA 代谢的所有步骤。它们是依赖于 ATP 的 RNA 结合蛋白和 RNA 依赖的 ATP 酶。它们可以置换短的双链体，但缺乏连续性。它们的机制和功能尚不清楚；经典或批量生化分析不足以回答这些问题。单分子技术提供了有用的工具，但在蛋白质非连续性和信号较弱的情况下，它们受到限制。我们在这里提出了一种新的、基于磁镊的单分子测定法，该方法简单，可以灵敏地测量一小段杂交 RNA 寡核苷酸的置换时间。同时可以分析数十个分子。比较有和没有解旋酶的置换时间可以深入了解蛋白质的酶活性。我们使用该测定法研究了酵母 Ded1，它与人 DDX3 同源。尽管 Ded1 作用于多种底物，但我们发现 Ded1 需要 RNA 底物才能进行其依赖于 ATP 的解旋活性，并且需要水解 ATP 才能观察到这种活性。此外，我们发现只有分子内单链 RNA 延伸增强了这种活性。我们提出了一个模型，其中 ATP 结合的 Ded1 稳定部分解开的双链体，并且可能需要多个结合事件才能观察到置换。
