Investigations into the mechanism for RNA structural remodeling by dead-box helicase proteins
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Structured RNAs and RNA-protein complexes (RNPs) are involved in many essential biological processes and the specific conformations of these RNAs are crucial to their various functions. However, in vitro studies have found that RNA has propensity for misfolding into inactive species that often consist of extensive secondary and tertiary interactions, which can be locally and globally stabilizing, resulting in long-lived non-native conformers. DEAD-box helicases are one class of proteins that have been found to accelerate folding and rearrangements of highly structured RNAs. While these proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt the tertiary interactions that often stabilize both native and misfolded RNA conformations. We used single molecule fluorescence to probe the mechanism by which DEAD-box proteins facilitate global unfolding of a structured RNA. DEAD-box protein CYT-19, a mitochondrial protein from Neurospora crassa, was found to destabilize a specific tertiary interaction with the Tetrahymena group I intron ribozyme using a helix capture mechanism. The protein molecule binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then uses ATP to unwind the helix, liberating the product strands. Ded1, a multi-functional DEAD-box protein found in Saccharomyces cerevisiae, 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 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.