Figure 6. Pre-rRNA fragments stimulate Dbp8 ATPase activity in vitro. (A) In vitro transcribed fragments of the 50ETS rRNA stimulate Dbp8 ATP hydrolysis in the presence of Esf2. ATP hydrolysis assays were carried out with 10 mM ATP, 10 pmol of Dbp8 and 10 pmol of Esf2, 1 mg/ml of total yeast RNA, 0.8 mM of ribosomal RNAs (schematically outlined on top of the graph) and 50 mM KCl. Samples were incubated at 30 C for 30 min. Plotted are the averages and standard errors derived from three independent experiments. (B) In vitro transcribed 50ETS rRNA fragments stimulate Dbp8 ATP hydrolysis in the absence of Esf2. ATP hydrolysis experiments were performed with 10 mM ATP, 5 pmol of Dbp8 and 50 mM KCl, in the presence or absence of equimolar amounts of Esf2 and various amounts of 50ETS rRNA (0–360 fragment; plotted on the X-axis). ATP hydrolysis (plotted on the Y-axis) was measured after 30 min incubation at 30 C. Plotted are the averages and standard errors derived from three independent experiments. amounts of the 50ETS 0-360 rRNA transcript was added to the reaction (Figure 6B). However, while rRNA was stimulat- ory for ATPase activity, the effect is small compared to the stimulatory effect of the Esf2 co-factor (30-fold under these conditions). DISCUSSION We have characterized the in vitro enzymatic activity of Dbp8, a DEAD box helicase required for small ribosomal subunit biogenesis (13). We identify the RRM-containing Nucleic Acids Research, 2006, Vol. 34, No. 10 3197 protein, Esf2, also required for small ribosomal subunit bio- genesis (19), as a Dbp8 binding protein that acts as an auxili- ary factor to stimulate Dbp8 ATPase activity. The C-terminal domain of Esf2 is required for Dbp8 binding, for stimulation of ATP hydrolysis and for its in vivo function. We show that Esf2 also binds promiscuously to RNA, and that while some rRNA fragments stimulate Dbp8 ATPase activity, Esf2 is a more potent stimulator of ATPase activity than RNA. We have thus shown that Esf2 is a co-factor for Dbp8 ATP hydrolysis. Our ﬁndings indicate that Dbp8 is typical of a DExD/H box RNA helicase with respect to its ATP hydrolysis activity [using Table 1 in Cordin et al. (6) as a basis for comparison]. It differs from most other DExD/H box RNA helicases char- acterized in vitro in that, under the conditions that we have used, its ATPase activity does not require added RNA. There is, however, a slight stimulation of ATPase activity when in vitro transcribed rRNA is added. Notably, of the ﬁve other DEAD box helicases required for ribosome biogen- esis that have been studied in vitro, three do not require RNA for ATPase activity (Dbp4, Dbp9 and Rok1), one requires it (Has1), and Rrp3 was not studied in enough detail (14–17,27). In contrast, the bacterial DExD/H box protein DbpA, also involved in ribosome biogenesis, has an absolute requirement for a speciﬁc portion of the 23S rRNA for in vitro ATP hydrolysis (28,29). Since DExD/H box RNA helicases generally participate in cellular processes that require RNA–protein complexes, it is not surprising that many DExD/H box helicases have known interacting proteins (9) and in some cases a single helicase can have multiple interacting proteins. New partners for cel- lular helicases continue to be identiﬁed, including ones from human viral pathogens, such as cytomegalovirus (CMV) (30). In general, protein co-factors are thought to increase the ATPase and unwinding activities of their associated helicase. They can also have other effects. For example, the interaction of the NS4A protein of hepatitis C virus with the viral DExD/ H helicase NS3 changes the helicase substrate speciﬁcity from DNA to RNA (31). In addition, an RNA helicase can act as an RNA chaperone, as in the case where the helicase CYT19 recruits CYT18 (tyrosyl-tRNA synthetase) to splice the group I intron in yeast mitochondria (32). However, of the studied interactions, there are only a handful of examples where the mechanism of the effect of the protein co-factor on helicase enzymatic properties has been elucidated in vitro. The eukaryotic translation factor DExD/H box RNA helicase eIF4A has several co-factors, of which eIF4B and eIF4G are the most well-studied. Both stimulate ATPase activity and RNA unwinding (33–37). As with the effect of Esf2 on Dbp8, addition of the eIF4B co- factor to the helicase eIF4A increases its afﬁnity for ATP (38). Similarly, RhlB, involved in RNA degradation as part of the E.coli degradosome, interacts with RNase E, which stimulates its ATPase activity (39). What is the mechanistic role of Esf2 in enhancing the ATPase activity of Dbp8? Esf2 binding to Dbp8 may stabilize the tertiary structure of Dbp8 or induce a conformational change that is optimal for Dbp8 activity. A similar mechan- ism has been proposed for the translation initiation factors eIF4A and its co-factor eIF4G, two proteins that are part of the eIF4F complex involved in recruiting the small ribosomal