Nucleic Acids Research, 2006, Vol. 34, No. 10 3189–3199 doi:10.1093/nar/gkl419 The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis Sander Granneman 1 , ChieYu Lin1, Erica A. Champion 2 , Madhusudan R. Nandineni1, Cornelia Zorca1 and Susan J. Baserga1,2,3,* 1Departments of Molecular Biophysics and Biochemistry, 2Genetics and 3Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA Received April 18, 2006; Revised May 2, 2006; Accepted May 19, 2006 ABSTRACT While 18 putative RNA helicases are involved in ribosome biogenesis in Saccharomyces cerevisiae, their enzymatic properties have remained largely biochemically uncharacterized. To better under- stand their function, we examined the enzymatic properties of Dpb8, a DExD/H box protein previously shown to be required for the synthesis of the 18S rRNA. As expected for an RNA helicase, we demon- strate that recombinant Dbp8 has ATPase activity in vitro, and that this activity is dependent on an intact ATPase domain. Strikingly, we identify Esf2, a nucleolar putative RNA binding protein, as a binding partner for Dbp8, and show that it enhances Dbp8 ATPase activity by decreasing the KM for ATP. Thus, we have uncovered Esf2 as the first example of a protein co-factor that has a stimulatory effect on a nucleolar RNA helicase. We show that Esf2 can bind to pre-rRNAs and speculate that it may function to bring Dbp8 to the pre-rRNA, thereby both regulating its enzymatic activity and guiding Dbp8 to its site of action. INTRODUCTION In eukaryotes, members of the family of DExD/H box RNA helicases are involved in many different steps of RNA meta- bolism, from RNA processing and RNA export to translation. The activity of RNA helicases is generally associated with the separation of RNA strands (‘unwinding’), hence the name RNA helicase. Recently, it has become evident that these proteins also have the capacity to anneal RNA strands or to remodel ribonucleoprotein (RNP) complexes by disrupt- ing protein–RNA interactions (1–4). Interestingly, the latter activity does not necessarily involve strand displacement (4). Thus, the term ‘RNA helicase’ is clearly a very simple description for the diverse biochemical properties of this family of proteins. DExD/H box RNA helicases generally harbor 7–9 con- served motifs, which comprise the core RNA helicase domain [reviewed in (5,6)]. These motifs are not only involved in binding of the RNA substrate, but also are required for bind- ing and hydrolysis of NTP, generally ATP, and use energy that is released in this reaction to drive the RNA/RNP remod- eling steps. The recently discovered Q motif, unique to DEAD box proteins, has been proposed to function in ATP and RNA substrate binding (7,8). Motifs I, II and VI are responsible for binding of ATP and the Mg2+ co-factor, whereas motif III (SAT) is implicated in coupling ATP hydrolysis with RNA unwinding activity. Because RNA helicases are involved in diverse steps in gene expression that require large RNA–protein complexes, it is likely that they are not acting alone but in association with other proteins. By the accounting of Silverman et al. in a recent review [Table 1 in (9)], there are at least 36 DExD/H box helicases that may have protein partners. Close inspection of this list indicates that only a few of the interactions have been characterized at the molecular level, and for even fewer have the biochemical parameters of the helicase been examined. Thus, the function of these putative co-factors in RNA helicase activity is largely unknown. Ribosome biogenesis in yeast involves the synthesis of a 35S polycistronic rRNA precursor (pre-rRNA) that is pro- cessed in large pre-ribosomal particles at several well-deﬁned sites to produce the mature 18S, 5.8S and 25S rRNAs [for a recent review see (10)]. This process requires over 200 pro- teins and hundreds of small nucleolar RNAs (snoRNAs), which function in cleavage and chemical modiﬁcation of pre-rRNA via complementary base pairing interactions with the rRNA. A total of 18 putative RNA helicases are involved in pre-rRNA processing and ribosome assembly. Mutational *To whom correspondence should be addressed at Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, SHM C-114, 06520-8024, USA. Tel: 203 785 4618; Fax: 203 785 6404; Email: susan.baserga@yale.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.