Sun Yingjie, Atas Evrim, Lindqvist Lisa M, Sonenberg Nahum, Pelletier Jerry, Meller Amit
Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia; Department of Biochemistry and The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC H3G 1Y6, Canada.
Structure. 2014 Jul 8;22(7):941-8. doi: 10.1016/j.str.2014.04.014. Epub 2014 Jun 5.
The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a "dumbbell" structure consisting of two domains connected by a flexible linker. Previous studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conformational dynamics of eIF4AI in real time using single-molecule FRET. We demonstrate that eIF4AI/eIF4H complex can repetitively unwind RNA hairpins by transitioning between an eIF4AI "open" and a "closed" conformation using the energy derived from ATP hydrolysis. Our experiments directly track the conformational changes in the catalytic cycle of eIF4AI and eIF4H, and this correlates precisely with the kinetics of RNA unwinding. Furthermore, we show that the small-molecule eIF4A inhibitor hippuristanol locks eIF4AI in the closed conformation, thus efficiently inhibiting RNA unwinding. These results indicate that the large conformational changes undertaken by eIF4A during the helicase catalytic cycle are rate limiting.
真核生物翻译起始因子4AI（eIF4AI）是典型的DEAD盒RNA解旋酶。它具有由柔性连接子连接的两个结构域组成的“哑铃”结构。先前的研究表明，eIF4AI与eIF4H一起结合到环结构上，并反复解开RNA发夹结构。在这里，我们使用单分子荧光共振能量转移实时探测eIF4AI的构象动力学。我们证明，eIF4AI/eIF4H复合物可以利用ATP水解产生的能量，通过在eIF4AI的“开放”和“封闭”构象之间转换，反复解开RNA发夹结构。我们的实验直接追踪了eIF4AI和eIF4H催化循环中的构象变化，这与RNA解旋动力学精确相关。此外，我们表明小分子eIF4A抑制剂马桑脂醇将eIF4AI锁定在封闭构象，从而有效抑制RNA解旋。这些结果表明，eIF4A在解旋酶催化循环中发生的大的构象变化是限速的。
