Iost I, Dreyfus M, Linder P
Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, 1211 Geneva 4, Switzerland.
J Biol Chem. 1999 Jun 18;274(25):17677-83. doi: 10.1074/jbc.274.25.17677.
The Ded1 protein (Ded1p), a member of the DEAD-box family, has recently been shown to be essential for translation initiation in Saccharomyces cerevisiae. Here, we show that Ded1p purified from Escherichia coli has an ATPase activity, which is stimulated by various RNA substrates. Using an RNA strand-displacement assay, we show that Ded1p has also an ATP-dependent RNA unwinding activity. Hydrolysis of ATP is required for this activity: the replacement of ATP by a nonhydrolyzable analog or a mutation in the DEAD motif abolishing ATPase activity results in loss of RNA unwinding. We find that cells harboring a Ded1 protein with this mutated DEAD motif are nonviable, suggesting that the ATPase and RNA helicase activities of this protein are essential to the cell. Finally, RNA binding measurements indicate that the presence of ATP, but not ADP, increases the affinity of Ded1p for duplex versus single-stranded RNA; we discuss how this differential effect might drive the unwinding reaction.
Ded1蛋白（Ded1p）是DEAD-box家族的成员之一，最近研究表明它对于酿酒酵母中的翻译起始至关重要。在此，我们发现从大肠杆菌中纯化得到的Ded1p具有ATP酶活性，该活性受到多种RNA底物的刺激。通过RNA链置换实验，我们表明Ded1p还具有ATP依赖的RNA解旋活性。此活性需要ATP水解：用不可水解的类似物替代ATP或DEAD基序中的突变消除ATP酶活性会导致RNA解旋活性丧失。我们发现携带这种突变DEAD基序的Ded1蛋白的细胞无法存活，这表明该蛋白的ATP酶和RNA解旋酶活性对细胞至关重要。最后，RNA结合测量表明，ATP的存在而非ADP的存在会增加Ded1p对双链与单链RNA的亲和力；我们讨论了这种差异效应如何驱动解旋反应。
