Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase.
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.
Iost
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Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, 1211 Geneva 4, Switzerland.
Dreyfus
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Linder
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002470:Cell Survival; D053487:DEAD-box RNA Helicases; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009711:Nucleotides; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C492152:DED1 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases