Poster abstracts

Poster number 42 submitted by Zhaofeng Gao

Two DEAD-box RNA helicases, eIF4A and Ded1p, function simultaneously in the eIF4F complex

Zhaofeng Gao (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Heath Bowers (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Andrea Putnam (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Xuan Ye (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106)

Abstract:
The eukaryotic translation initiation factor 4F (eIF4F) contains the large scaffold protein eIF4G, the cap-binding protein eIF4E, and the loosely associated DEAD-box RNA helicase eIF4A. In Saccharomyces cerevisiae, another DEAD-box RNA helicase Ded1p also interacts with eIF4G. To understand how the Ded1p-eIF4F interaction impacts the molecular function the proteins, we examined the consequences of this interaction on the biochemical activities of the eIF4F components and of Ded1p, using reconstituted complexes. We show that Ded1p allows eIF4G/eIF4E to associate with short RNAs that eIF4G/eIF4E alone cannot bind, and that Ded1p and eIF4G/eIF4E facilitate each other's binding to longer RNAs. eIF4G/eIF4E enhances the RNA-stimulated ATP hydrolysis by Ded1p, and establishes the most stable interaction with Ded1p when the helicase is bound to RNA and ATP. At the same time, eIF4G/eIF4E inhibits RNA unwinding by Ded1p by interfering with oligomerization of Ded1p, which is needed for optimal unwinding activity. However, addition of eIF4A to the eIF4G/eIF4E complex stimulates unwinding activity by Ded1p, even though eIF4A contributes only negligible unwinding activity on its own. Nonetheless, this stimulation requires ATP hydrolysis by eIF4A. We also show that Ded1p enhances the binding of eIF4E to the RNA 5’-cap, but only in the presence of eIF4G. Collectively, our results show that the Ded1p-eIF4F interaction impacts multiple biochemical properties of Ded1p and eIF4F, and that this modulation requires two functional DEAD-box helicases. This synergism suggests that both eIF4A and Ded1p can work simultaneously in conjunction with eIF4G and eIF4A.

Keywords: DEAD-box helicase, eIF4F, Ded1p