3190 Nucleic Acids Research, 2006, Vol. 34, No. 10 studies of RNA helicases involved in ribosome biogenesis revealed that conserved amino acids predicted to be involved in ATP binding and hydrolysis are important for cell viability, suggesting that they may indeed function as RNA helicases (11–13). Thus far, only four of the 18 proteins (Dbp4, Rok1, Dbp9 and Has1) have been characterized in vitro and have been shown to harbor ATPase activity (14–17). Two of these proteins (Dbp9 and Has1) were also able to separate artiﬁcial nucleic acid duplexes in vitro, suggesting that they are indeed helicases (14,17). However, biochemical evidence to support this hypothesis for the other 14 helicases is lacking. We have begun to study the enzymatic properties of the RNA helicases involved in ribosome biogenesis in order to better understand their function. Here we present the bio- chemical characterization for the yeast DEAD box protein Dbp8, a putative RNA helicase involved in 18S rRNA syn- thesis (13). We demonstrate that puriﬁed recombinant Dbp8 exhibits ATPase activity in vitro, and that mutations in the predicted ATPase domain abrogate Dbp8 ATPase activity. Strikingly, we identify Esf2, a nucleolar RNA binding protein required for pre-18S rRNA synthesis (18,19), as a Dbp8- interacting protein. Using two-hybrid technology and with puriﬁed recombinant proteins, we show that Esf2 does indeed directly bind to Dbp8, likely via its C-terminal domain, and that this interaction substantially increases its ATPase activ- ity. Thus, optimal ATP hydrolysis activity of Dbp8 depends on the activity of its protein co-factor, Esf2. Our results sug- gest a model in which Esf2 recruits Dbp8 to pre-ribosomes by binding to a region within the pre-rRNA. To our knowledge, Dbp8 is only the second known eukaryotic RNA helicase that requires a protein co-factor for optimal function. MATERIALS AND METHODS Yeast strains and media YPH499 (mata, ura3-52, lys2-80, ade2-101, trp1-D63, his3-D 200, leu2-D1) was the parental strain for the construction of strains used here. Strains carrying genes under the control of the galactose-inducible promoter or strains carrying triple HA-tagged genes (KanR marker) were generated as described previously (20). Strains expressing TAP carboxyl-tagged Dbp8 (Kluyveromyces lactis TRP marker) were generated as described previously (21). Unless speciﬁcally stated, strains were grown in YPD (1% yeast extract, 2% peptone and 2% dextrose) and GAL::3HA strains were grown in YPG/R (1% yeast extract, 2% peptone, 2% galactose and 2% rafﬁnose) at 30 C. Strains harboring p415GPD::ESF2 plasmids (CEN origin of replication, LEU marker) were grown at 30 C in SC—leucine (Clontech), supplemented with 2% dextrose or with 2% galactose and 2% rafﬁnose. DNA manipulations and in vitro RNA transcriptions To over-express and purify six histidine-tagged Dbp8 protein, DBP8 was ampliﬁed by PCR from genomic DNA and cloned into the pET28a expression vector using BamHI and NotI restriction sites. To over-express and purify glutathione S-transferase GST-Esf2, GST-Esf2 DC and GST-Rpa34, the alleles were ampliﬁed by PCR using genomic DNA as template and the products were cloned into the pGEX6P-2 vector using BamHI, EcoRI and XhoI restriction sites. All constructs were veriﬁed by automated DNA sequencing (W.M. Keck DNA sequencing facility at Yale). For the yeast 2-hybrid experiments ESF2 was PCR ampliﬁed and cloned into the pAS2-1 bait vector using NcoI and BamHI restriction sites. To clone DBP8 into the prey vector (pACT2), the gene was ampliﬁed by PCR and cloned into pACT2 using SmaI and BamHI restriction sites. To generate in vitro transcribed rRNA fragments, rDNA templates containing a T7 promoter were constructed by PCR using yeast genomic DNA. The rRNA transcripts were generated using the MEGAscript kit according to the manufacturer’s instructions (Ambion). Expression and purification of recombinant proteins The fusion proteins were expressed in Escherichia coli BL21 (DE3). Cells were grown in Luria–Bertani (LB) at 37 C sup- plemented with either kanamycin (pET28a) or ampicillin (pGEX) to an OD600 of 0.4 and then grown for 1 h at 17 C. Protein expression was induced with 0.5 mM isopropyl b-D- thiogalactoside (IPTG) for 16–20 h at 17 C. Cells were harvested by centrifugation and pellets were resuspended in 5 vol of breaking buffer [150 mM KCl, 50 mM Tris–HCl (pH 7.4), 10% sucrose, 0.5 mM EDTA, 1 mM b- mercaptoethanol, 0.01% Igepal (Sigma)] containing a protease inhibitor cocktail (10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml chymostatin, 10 mg/ml pepstatin, 1.3 mM benzamidine; Sigma) and lysed using a Constant sys- tems cell disrupter (Low March, Daventry Northants, UK) at 20 000 psi (1350 Bar). Extracts were clariﬁed by ultracentri- fugation in a Ti45 rotor at 100 000 g for 1 h. To purify recom- binant six histidine-tagged (His6) Dbp8 proteins, clariﬁed extracts prepared from 25 g of cells was passed over a 30 ml SP Sepharose column pre-equilibrated with K100 buf- fer (like K1000 but with 100 mM KCl instead of 1 M). The SP Sepharose column was subsequently washed with 3 vol of K100 and bound proteins were eluted with an 80 ml linear salt gradient (100–1000 mM KCl) prepared in K-buffer [20 mM KHPO4 (pH 7.4), 0.5 mM EDTA, 10% glycerol, 1 mM b-mercaptoethanol and 0.01% Igepal], followed by a 20 ml K1000 elution. Fractions enriched for His6-Dbp8 were pooled and the mixture was then incubated with 5 ml of Ni-NTA beads (Qiagen; 1 ml beads / 5 g of cell pellet) for 1 h at 4 C. The beads were then washed with 10 vol of K1000 followed by a wash with 10 vol of K300 containing 50 mM imidazole. Proteins were eluted in K300 containing 250 mM imidazole. Fractions containing nearly homogenous recombinant proteins were pooled and dialyzed for 4 h in K300 buffer containing 20% glycerol at 4 C. To purify the GST-Esf2 wild-type and Esf2 DC fusion pro- teins, clariﬁed extracts prepared from 10 g of cells were passed over 30 ml Heparin agarose columns (BioRad) and the fusion proteins were eluted by applying an 80 ml linear salt gradient (100–2000 mM KCl) prepared in K-buffer, fol- lowed by 20 ml K2000 elution step. Fractions enriched for the fusion proteins were pooled ([KCl] 300 mM) and incub- ated with 4 ml of glutathione–Sepharose beads (Amersham) overnight at 4 C. After extensively washing the beads with K300 buffer, the proteins were eluted in K300 containing