Ded1p, a DEAD-box Protein Required for Translation Initiation in Saccharomyces cerevisiae, Is an RNA Helicase*
- From the ‡Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, 1211 Geneva 4, Switzerland and the ¶Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 75230 Paris, France
Abstract
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 duplexversus single-stranded RNA; we discuss how this differential effect might drive the unwinding reaction.
Footnotes
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↵* This work was supported by grants from the Swiss National Science Foundation (to P. L.) and Grant 1341 from the Association pour la Recherche sur le Cancer (to M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported by a long-term fellowship from EMBO and by the Helmut Horten Foundation. To whom correspondence should be addressed: Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris, Cedex 05, France.
- Abbreviations:
- ss
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single-stranded
- ds
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double-stranded
- AMP-PNP
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5′-adenylyl imidodiphosphate
- YCp
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yeast centromeric plasmid
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- Received February 4, 1999.
- Revision received April 12, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
