Abstract DEAD-box helicases are essential for gene expression and RNA metabolism. However, the mechanisms regulating their activity remain largely elusive. The DEAD-box helicase DDX39B/UAP56 forms a 2:1 complex with the C-terminal domain (CTD) of RNA-binding protein Tho1, but the functional relevance of this interaction is still elusive. Here, we show that the Tho1-CTD stimulates the helicase activity of Sub2, the yeast homologue of DDX39B/UAP56, by acting as a rigid scaffold that promotes Sub2 oligomerization on RNA. The Tho1-CTD has two conserved α-helical motifs, each interacting with one Sub2, and we demonstrate that both motifs are essential for the stimulation. This scaffolding mechanism is shared across species, as the Tho1 ortholog MOS11 from Arabidopsis thaliana stimulates A. thaliana UAP56. Interestingly, MOS11 has five of the conserved α-helical motifs, which are connected by flexible linkers. We show that the number and spatial separation of these motifs are critical for stimulation and that MOS11 stimulates unwinding on a broader range of substrates than the Tho1-CTD. The cofactor-mediated helicase oligomerization is reminiscent of the self-oligomerization observed for other DEAD-box helicases. Furthermore, our data illustrate how cofactor architecture affects substrate specificity and provide a comprehensive mechanistic framework for cofactor-mediated helicase activation.
Becker et al. (Fri,) studied this question.