title: Single-molecule kinetics of the eukaryotic initiation factor 4AI upon RNA unwinding.
abstract: 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.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
authors.forename: Evrim
authors.initials: E
authors.lastname: Atas
authors.affiliation: 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.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Lindqvist
authors.affiliation: Department of Biochemistry and The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC H3G 1Y6, Canada.
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
authors.affiliation: Department of Biochemistry and The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC H3G 1Y6, Canada.
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA; Faculty of Biomedical Engineering, The Technion, Haifa 32000, Israel. Electronic address: ameller@bu.edu.
authors.forename: Amit
authors.initials: A
authors.lastname: Meller
mesh_terms: D000818:Animals; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012313:RNA; D039601:Eukaryotic Initiation Factor-4A; D053487:DEAD-box RNA Helicases