Nucleic Acids Research, 2006, Vol. 34, No. 10 3193 Figure 2. Dbp8 motif I and II mutants are defective in ATP hydrolysis in vitro. (A) Schematic representation of conserved motifs in Dbp8. The mutations that were introduced in motifs I and II are indicated by arrows. (B) SDS–PAGE analysis of purified His6-Dbp8 motif I and II mutants. Two mg of each purified protein was resolved by SDS–PAGE. Proteins were either stained with Coomassie brilliant blue (left panel) or subjected to western blot analysis (right panel) using anti-His6 antibodies. (C) Motif I and II mutants are ATPase defective. ATP hydrolysis assays were performed with 10 mM ATP, in the presence of 300 mM KCl and using 10 pmols of Dbp8 wild-type or motif I and II mutant proteins (X-axis). Mixtures were incubated for 30 min at 30 C and 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 To substantiate the in vivo interaction, we asked whether recombinant Dbp8 and Esf2 would directly interact in vitro (Figure 3B). Esf2, expressed and puriﬁed as a GST-fusion protein from bacterial extracts, was incubated with equimolar amounts of His6-Dbp8. Reconstituted complexes were precipitated using glutathione–Sepharose beads and bound proteins were resolved by SDS–PAGE and stained with Coomassie brilliant blue. The results show that GST-Esf2 co-precipitated approximately stoichiometric amounts of Dbp8 (Figure 3B, lane 2). As negative controls we included GST alone, and two other recombinant proteins: His6-tagged Fap7 and GST-Rpa34 (Figure 3B, lanes 4–12). Fap7 is involved in 20S pre-rRNA processing but is not required for earlier processing steps and is therefore used as a negative control (24). Dbp8 was shown to co-purify with TAP-tagged Rpa34 (a subunit of RNA polymerase I) in afﬁnity puriﬁca- tion experiments (25) and GST-Rpa34 was therefore included to test a potentially direct interaction with His6-Dbp8. The results show that Esf2 (predicted pI 8.4) did not co-precipitate His6-Fap7 (pI 4.4; Figure 3B, lane 5). Furthermore, GST alone and GST-Rpa34 (pI 9.5) did not interact with Dbp8 (pI 10) in vitro (Figure 3B, lanes 8 and 11), suggesting that the observed interactions between Esf2 and Dbp8 are not solely the result of non-speciﬁc elec- trostatic interactions. We conclude that Dbp8 directly and speciﬁcally binds Esf2 in vitro. Finally, to conﬁrm again if these proteins associate in vivo we performed co-immunoprecipitation experiments using strains in which Dbp8 was TAP-tagged and Esf2 was tagged with three copies of the HA tag (3HA). Extracts prepared from these strains were incubated with IgG Sepharose beads to immunoprecipitate Dbp8-TAP and co-precipitated proteins were analyzed by SDS–PAGE and western blot analysis using anti-HA antibodies (Figure 3C). We found that a small fraction of HA-tagged Esf2 associated with TAP-tagged Dbp8 in yeast extracts (Figure 3C, lane 6). As a positive control we used a strain in which two 66S pre- ribosome associated proteins were tagged (Rpf2-TAP, Rpf1-3HA; Figure 3C, lanes 1 and 2). As a negative control we used a strain in which only Esf2 was 3HA-tagged (Figure 3C, lanes 3 and 4). Thus, Esf2 directly binds Dbp8 in vitro and is complexed with Dbp8 in vivo.