Single-molecule fluorescence experiments reveal how DEAD-box proteins unfold structured RNAs to promote conformational transitions and refolding to the native functional state.
DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome.
In addition to carrying genetic information from DNA to protein, RNAs function in many essential cellular processes. This often requires the RNA to form a specific three-dimensional structure, and some functions require cycling between multiple structures. However, RNAs have a strong propensity to become trapped in nonfunctional, misfolded structures. Due to the intrinsic stability of local structure for RNA, these misfolded species can be long-lived and therefore accumulate. ATP-dependent RNA chaperone proteins called DEAD-box proteins are known to promote native RNA folding by disrupting misfolded structures. Although these proteins can unwind short RNA helices, the mechanism by which they act upon higher order tertiary contacts is unknown. Our current work shows that DEAD-box proteins capture transiently exposed RNA helices, preventing any tertiary contacts from reforming and potentially destabilizing the global RNA architecture. Helix unwinding by the DEAD-box protein then allows the product RNA strands to form new contacts. This helix capture mechanism for manipulation of RNA tertiary structure does not require a specific binding motif or structural environment and may be general for DEAD-box helicase proteins that act on structured RNAs.