Granneman Sander, Lin ChieYu, Champion Erica A, Nandineni Madhusudan R, Zorca Cornelia, Baserga Susan J
Department of Molecular Biophysics, Yale University School of Medicine, New Haven, Connecticut 06520-8024, USA.
Nucleic Acids Res. 2006 Jun 13;34(10):3189-99. doi: 10.1093/nar/gkl419. Print 2006.
While 18 putative RNA helicases are involved in ribosome biogenesis in Saccharomyces cerevisiae, their enzymatic properties have remained largely biochemically uncharacterized. To better understand 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 demonstrate 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 K(M) 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.
虽然18种假定的RNA解旋酶参与酿酒酵母的核糖体生物合成，但其酶学特性在很大程度上仍未得到生化表征。为了更好地理解它们的功能，我们研究了Dpb8的酶学特性，Dpb8是一种DExD/H盒蛋白，先前已证明其是18S rRNA合成所必需的。正如对RNA解旋酶的预期，我们证明重组Dbp8在体外具有ATP酶活性，且该活性依赖于完整的ATP酶结构域。令人惊讶的是，我们鉴定出Esf2，一种核仁假定的RNA结合蛋白，作为Dbp8的结合伴侣，并表明它通过降低ATP的米氏常数（Km）来增强Dbp8的ATP酶活性。因此，我们发现Esf2是对核仁RNA解旋酶具有刺激作用的蛋白质辅因子的首个例子。我们表明Esf2可以结合前体rRNA，并推测它可能起到将Dbp8带到前体rRNA的作用，从而既调节其酶活性又将Dbp8引导至其作用位点。
