3192 Nucleic Acids Research, 2006, Vol. 34, No. 10 Figure 1. Purified recombinant Dbp8 has ATPase activity. (A) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). (B–D) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42 C), different pH (C) and varying potassium chloride concentration (D) with 10 mM ATP. ATP conversion (Y-axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments. either Coomassie blue staining or western blot analysis using an anti-six histidine antibody (Figure 2B). Little or no degradation products could be detected by either method and the mutations did not appear to dramatically affect the accumulation or stability of the protein (Figure 2B). Com- parison of the ATPase activities of the wild-type and mutant proteins indicated that wild-type Dbp8 protein hydrolyzed signiﬁcantly higher amounts of ATP than the Motif I (K52A and K52R) and Motif II mutants (D157A) (Figure 2C). Under the tested conditions, the D157A mutant had 2% of the wild-type activity, whereas the Walker A mutations reduced ATPase activity roughly 10-fold. Thus, we conclude that the invariant Motif I lysine and the ﬁrst aspartic acid in the Motif II are not only essential for cell viability but also are required for Dbp8 ATP hydrolysis activity in vitro. Dbp8 directly interacts with pre-rRNA processing protein Esf2 Other investigators had previously found that Dbp8 interacts with Esf2 in a large-scale yeast 2-hybrid screen (18). Esf2 is a nucleolar RRM-containing protein, which, like Dbp8, is required for processing at sites A0, A1 and A2 in the pre- rRNA (19). The 2-hybrid interaction and the similar function in pre-rRNA processing suggested that they might function together. We have conﬁrmed the predicted in vivo interaction through a directed yeast 2-hybrid assay (Figure 3A).