200 mM reduced glutathione (USB). Fractions containing nearly homogenous recombinant proteins were pooled and extensively dialyzed in K300 buffer containing 20% glycerol at 4 C. Proteins were frozen in small aliquots in liquid nitro- gen and stored at 80 C. The purity of the puriﬁed material was around 90% as judged by SDS–PAGE and Coomassie blue staining. Protein concentrations were determined using the Bradford assay as described by the manufacturer (BioRad). ATPase experiments Unless otherwise noted, ATPase assays were carried out using 10 pmol of recombinant His6-Dbp8 in a Tris-based buf- fer (pH 7.5) containing 5 mM MgCl2, 1 mM DTT, 1 mg BSA and 10 mM cold ATP (pH 7.0, Sigma) in a ﬁnal reaction vol- ume of 10 ml. Radioactive 32P-g-ATP was added as a tracer in order to quantify the amount of ATP converted to ADP. Reaction products were separated by thin layer chromato- graphy and visualized via autoradiography. The ATP conver- sion rate was determined by quantifying 32P release using a phosphorimager. In vitro protein–protein and protein–RNA interactions For the protein–protein interaction assays, 300 pmol of each protein was mixed in a K0 buffer [20 mM KHPO4 (pH 7.4), 1 mM DTT and 0.1% Igepal] in a ﬁnal volume of 100 ml. Unless otherwise noted, the potassium chloride concentration in the pull-down reactions was 150 mM. After 1 h incubation on ice, 100 ml of K150 containing 0.1% Igepal was added to the mixtures and 20 ml was removed for analysis (10% input). The remainder (180 ml) was subsequently incubated with 20 ml of glutathione–Sepharose beads (Amersham) for 2 h at 4 C. After this incubation step, 18 ml of the supernatant was removed for analysis (10% supernatant). The beads were subsequently washed three times with 500 ml of ice- cold K150 containing 0.1% Igepal. Bound proteins were eluted by boiling the beads in SDS–PAGE loading dye and resolved by 10% SDS–PAGE. Proteins were visualized by Coomassie brilliant blue staining. For the protein–RNA interaction assays, 10 pmol of GST- fusion protein (diluted in K150) was incubated with 10 ng of in vitro transcribed and 32P-labeled rRNA fragments in K0 buffer in a ﬁnal volume of 10 ml. KCl was added to a ﬁnal concentration of 150 mM. After 1 h incubation on ice, K150 was added to a ﬁnal volume of 200 ml, 20 ml of the sample was removed (10% input) and the remainder (180 ml) was incubated with 20 ml of glutathione–Sepharose beads (Amersham) for 1 h on ice with regular agitation. The beads were subsequently washed three times with 500 ml of ice-cold K150 containing 0.1% Igepal. Beads were then resuspended in 300 ml K150 buffer and RNA was isolated by phenol–chloroform–isoamylalcohol extraction and ethanol precipitation. Co-precipitated RNAs were resolved on 6% polyacrylamide-8 M urea gels and visualized by autoradiography. Miscellaneous Yeast 2-hybrid, immunoprecipitation experiments and western blot analyses were performed as described else- where (22). Antibodies used: horseradish peroxidase (HRP) Nucleic Acids Research, 2006, Vol. 34, No. 10 3191 conjugated mouse anti-His6 (Sigma). The 12CA5 anti-HA monoclonal antibody has been described elsewhere (22). Motif searches for Esf2 were carried out with Interpro (http://www.ebi.ac.uk/interpro/), SMART (http://smart. embl-heidelberg.de/), Prosite (http://us.expasy.org/prosite/) and the COILS program (http://www.ch.embnet.org/ software/COILS_form.html), (23). RESULTS Recombinant Dbp8 has ATPase activity The presence of the canonical DEAD box motifs in Dbp8 suggests that it may function as an ATPase and possess RNA unwinding activity. Recent studies have demonstrated that mutations at conserved amino acids within the putative ATPase motifs of Dbp8 are lethal, demonstrating that these motifs are essential for Dbp8 function in vivo (12,13). More- over, over-expression of these mutants in a wild-type back- ground causes dominant negative growth defects and delays in pre-rRNA processing as well as 18S rRNA synthesis (12,13). These combined results suggested that Dbp8 has ATP hydrolysis activity and that, furthermore, this activity is critical for 18S rRNA synthesis. However, biochemical evidence in support of this hypothesis was lacking. Thus, to gain more detailed insight into the biochemical properties of Dbp8 and its role in 18S rRNA synthesis, we have ana- lyzed the ATPase activity of recombinant Dbp8 proteins in vitro. To substantiate the in vivo results, we expressed and puriﬁed a six histidine-tagged Dbp8 protein (His6-Dbp8) from E.coli lysates (Figure 1A) and characterized its ATPase activity under a variety of conditions (Figure 1B–D). Our results indicated that Dbp8 had a strong preference for Mg2+ as metal co-factor (data not shown), was most active at 30 C (Figure 1B), had a pH optimum for activity of 7.4 (Figure 1C) and a salt optimum of 300 mM KCl (Figure 1D). Much to our surprise, under the tested condi- tions, Dbp8 ATPase activity was not signiﬁcantly stimulated by the presence of total yeast RNA, single-stranded or double-stranded DNA (data not shown). The presence of an excess of RNAse A in the ATPase assay did not noticeably affect Dbp8 ATP hydrolysis activity and Dbp8 puriﬁed from RNAse A treated E.coli extracts showed signiﬁcant ATP hydrolysis activity (data not shown). Thus, under the tested conditions (pH 7.4, 300 mM KCl, 30 C, 5 mM MgCl2) Dbp8 ATP hydrolysis activity does not require an RNA co-factor. Conserved amino acids within the ATPase motifs are essential for Dbp8 ATPase activity in vitro Mutations in the putative ATPase motifs (Motif I and II; K52A, K52R and D157A) are lethal in vivo and, when over-expressed, delay pre-rRNA processing and synthesis of the 18S rRNA (12,13). To correlate these in vivo results with the biochemical properties of Dbp8, we addressed whether these mutated proteins were capable of hydrolyzing ATP in vitro (Figure 2A). Mutant Dbp8 proteins were expressed and puriﬁed as described above and the quality of the protein preparations was determined by SDS–PAGE followed by