Ded1p, a DEAD-box Protein Required for Translation Initiation in Saccharomyces cerevisiae, Is an RNA Helicase

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 duplex versus singlestranded RNA; we discuss how this differential effect might drive the unwinding reaction.DEAD-box proteins form a large family of putative RNA helicases that show sequence similarity to eIF4A, a eukaryotic translation initiation factor. All members of the DEAD-box family share eight conserved amino acid motifs, including the characteristic sequence Asp-Glu-Ala-Asp (DEAD in the singleletter code) that inspired their name (1). Sequence comparisons identified the closely related DEAH-and DExH-box families, which together with the DEAD-box proteins, form the helicase superfamily II (2). The DEAH and DExH families notably include DNA helicases involved in DNA replication and recombination. The putative RNA helicases of superfamily II are found in a wide range of organisms, including bacteria, viruses, and eukaryotes ranging from yeast to humans. Although they are involved in very diverse cellular functions, such as pre-mRNA splicing, rRNA processing, and mRNA export, translation, and decay, they are all supposed to share in common an RNA helicase activity (3, 4). This activity has been inferred from the ability of eIF4A to melt out mRNA structure (5) or to dissociate an RNA duplex in vitro (6), in an ATP-dependent manner. However, although NTPase activity has been demonstrated for all purified DEAD-box and related proteins, RNA helicase activity has been characterized for only a few of them and remains conjectural in most cases.Nevertheless, numerous steps of gene expression are likely to require RNA helicase activity, either to unwind RNA secondary structures or to rearrange large RNA structures, or even to disrupt RNA-protein interactions. For example, transient base pairings between small nuclear RNAs and between small nuclear RNAs and pre-mRNA, which occur during pre-mRNA splicing, are often mutually exclusive and thus need to form and dissociate sequentially. At least eight DEAD-box and related proteins have thus far been shown to be required for splicing in yeast and may accomplish these structural rearrangements (7). Similarly, 13 DEAD-box are assumed to be involved in extensive rearrangements between pre-rRNA and ribos...

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…Consistent with these observations, kinetic evidence suggests that phosphate release might be linked to RNA release (Henn et al, 2008). Both findings are consistent with the reduced ssRNA affinity of DEAD box proteins in complex with ADP (Lorsch and Herschlag, 1998a;Iost et al, 1999;Cordin et al, 2004). …”

“…Examples are the splicing helicases Cyt-19 and Mss116 (Mohr et al, 2002;Huang et al, 2005;Halls et al, 2007) that are general RNA chaperones, and Ded1p, a helicase involved in translation (Iost et al, 1999). These proteins bind unspecifically to structured RNA via their basic Cterminal extensions, and their helicase cores then unwind loosely attached neighboring double helical regions (Grohman et al, 2007;Mohr et al, 2008).…”

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

“…Our demonstration that NOH61 exhibits the capacity to hydrolyze ATP is not surprising, because many proteins that possess the superfamily II helicase domain have been shown previously to be ATPases. However, NOH61 ATPase activity displayed only a modest stimulation (1.6-to 3.9-fold) in the presence of polynucleotides, in apparent contrast to other DEAD-box proteins (for example, see proteins Dbp5p [Tseng et al, 1998] and Ded1p [Iost et al, 1999]). Nevertheless, it should be noted that there are several viral proteins whose ATPase activity is stimulated only weakly by nucleic acids (Kadare and Haenni, 1997).…”

From unwinding to clamping — the DEAD box RNA helicase family

“…Similar structures with ATP ground and transition state analogues are also consistent with the notion that these conformations represent states after the strand separation event 13 . ADP reduces the affinity of DEAD box proteins for RNA, and thus promotes dissociation of the protein from the substrate 46 .…”