3194 Nucleic Acids Research, 2006, Vol. 34, No. 10 Esf2 directly binds rRNA in vitro Esf2 contains a predicted RRM RNA binding domain, and we therefore examined whether Esf2 would directly bind RNA (19). Using in vitro transcribed rRNA fragments ( 200– 300 nt in length), we sought to determine if GST-Esf2 binds RNA in vitro. As TAP-tagged Esf2 associates with the 50ETS of the pre-rRNA in vivo (19), we generated two radiolabeled rRNA transcripts that largely cover the 50ETS region (Figure 3D). In addition, we made transcripts that con- tained 18S and 25S rRNA coding sequences (Figure 3D). Recombinant GST-Esf2 and GST alone (used as a negative control) were incubated with the various rRNA transcripts on ice to allow binding to occur and reconstituted complexes were precipitated using glutathione–Sepharose beads. Bound rRNAs were resolved by denaturing PAGE and detected by autoradiography. The results demonstrate that GST-Esf2 is able to bind all the tested rRNA fragments although less efﬁ- ciently to the 50ETS 361–799 rRNA fragment (Figure 3D, lane 2). Furthermore, GST alone did not interact with any of the tested rRNA fragments (Figure 3D, lane 5). We con- clude that recombinant GST-Esf2 binds RNA in vitro, albeit likely without any apparent sequence speciﬁcity. Direct binding of Esf2 to Dbp8 stimulates Dbp8 ATPase activity in vitro likely by increasing its affinity for ATP Since the two proteins interact, we asked whether Esf2 inter- action with Dbp8 would stimulate its ATPase activity. We performed ATPase assays in which increasing amounts of Esf2 were added to the reactions, up to 1.5 M excess over His6-Dbp8 (Figure 4A). Because we initially could not detect a signiﬁcant stimulation of ATPase activity by Esf2 in buffers containing 300 mM KCl, we performed the reactions with 200 mM KCl and indeed found a modest but signiﬁcant increase in ATP hydrolysis with increasing Esf2. Under these conditions, addition of 1.5 M excess of GST-Esf2 to the reaction stimulated Dbp8 ATPase activity 2-fold (Figure 4A). In contrast, the presence of 1.5 M excess of GST-Rpa34 did not have a stimulatory effect on Dbp8 ATPase activity. GST-Esf2 and GST-Rpa34 alone showed only residual background activity, usually consisting of hydrolyzing 1–5% of the total ATP. For comparison, back- ground hydrolysis of ATP in buffer during the reaction times was usually about 1 or 2% (data not shown). Surprisingly, when we analyzed Dbp8 ATPase activity in the presence of GST-Esf2 over varying KCl concentrations (50–450 mM; Figure 4B), the highest Dbp8 ATPase activity Figure 3. Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro. (A) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media ( His selection). (B) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 (24). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. (C) Esf2 associates with Dbp8 in vivo. Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4 C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. (D) GST-Esf2 directly binds RNA in vitro. GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 50ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione– Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.