3196 Nucleic Acids Research, 2006, Vol. 34, No. 10 binding to Dbp8 is required for its stimulatory effect, and the C-terminal domain of Esf2 is required for Dbp8 binding. Finally, we determined whether the mutant Esf2 proteins were functional in vivo. Since Esf2 is essential for viability, we tested whether the deletion mutants could support growth using a strain where the endogenous protein was under the control of a galactose-inducible/dextrose repressible promoter (GAL::3HA-ESF2). Serial dilutions of cells expressing the wild-type or mutant Esf2 proteins or carrying empty vector (p415GPD) were spotted on galactose based solid medium (to verify even spotting of the culture) and plates containing dextrose media (to deplete endogenous Esf2). The results show that the Esf2 DC and DRRM mutants were unable to support growth in cells depleted of endogenous Esf2 (Figure 5D). Interestingly, the N-terminal deletion did not appear to dramatically affect Esf2 function, since this mutant alleviated the growth defect in Esf2-depleted cells as well as the wild-type protein expressed from the plasmid. Collectively, these results suggest that the C-terminal domain of Esf2 is essential for function, required for binding to Dbp8 and for stimulation of ATPase activity. In contrast, the N-terminal region ﬂanking the putative RRM motif is neither required for Esf2 function nor binding to Dbp8. 50ETS rRNA fragments stimulate Dbp8 ATPase activity in vitro The association of Esf2 with the 50ETS RNA in vitro and in vivo [this work, (19)], prompted us to investigate whether pre-rRNAs stimulate Dbp8 ATPase activity. We tested total yeast RNA (type III; Sigma) and the four rRNA transcripts that bound to GST-Esf2 in the in vitro binding studies (Figure 3D). We found only a small increase in Dbp8 ATPase activity ( 1.35-fold; Figure 6A) at 50 mM KCl with equimo- lar amounts GST-Esf2 and Dbp8 (1 mM) in the presence of the 50ETS rRNAs (0.8 mM; 1–360, 361–799; Figure 6A). In contrast, total yeast RNA (1 mg/ml), the 18S rRNA and the 25S rRNA transcripts did not have any signiﬁcant stimulatory effect under the tested conditions. To corroborate these results we performed titration experiments to determine the concentration of 50ETS 0-360 RNA required for optimal Dbp8 ATPase activity and to test whether stimulation was dependent on the Esf2 co-factor (Figure 6B). Surprisingly, the best degree of stimulation by rRNA (6-fold under these conditions) occurred in the absence of Esf2 when equimolar Figure 5. The essential C-terminal domain of Esf2 is required for binding to Dbp8 and stimulating its ATPase activity in vitro. (A) Schematic representation of the Esf2 wild-type (WT) and deletion mutants (DN, DC and DRRM) that were tested in the yeast two-hybrid screen for their association with Dbp8. The predicted RRM motif and coiled-coil domains are represented as boxes. The amino acid positions relevant to the deletions made are indicated. (B) The C-terminal domain of Esf2 is required for Dbp8 interaction in the yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying either the Esf2 wild-type or Esf2 deletion mutants (bait) in combination with either Dbp8 (prey) or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (His selection). (C) The C-terminal domain of Esf2 is required for stimulation of Dbp8 ATPase activity in vitro. ATP hydrolysis experiments were performed with 10 mM ATP, 5 pmol of Dbp8 and 50 mM KCl, in the presence or absence (Dbp8 alone) of 5 pmols of GST-Esf2 wild-type (WT) or GST-Esf2 DC. ATP hydrolysis (plotted on the Y-axis) was measured after 30 min incubation at 30 C. Graphed are the averages and standard errors derived from three independent experiments. (D) The C-terminal domain of Esf2 is essential for function in vivo. Serial dilutions (10-fold) of GAL::3HA-ESF2 strains carrying the empty vector or, p415GPD-ESF2 wild-type and mutant alleles (DN, DRRM and DC) were grown in synthetic galactose media (SG/R-LEU) and spotted on either galactose containing plates (left panel; SG/R-LEU) or galactose containing plates (right panel; SD-LEU).