Local and global investigations into DEAD-box protein function

Numerous essential cellular processes, such as gene regulation and tRNA processing, are carried out by structured RNAs. While in vitro most RNAs become kinetically trapped in non-functional misfolded states that render them inactive on a biologically-relevant time scale, RNAs folding in vivo do not share this same outcome. RNAs do indeed misfold in the cell; however, chaperone proteins promote escape from these non-native states and foster folding to functional conformations. DEAD-box proteins are ATP-dependent RNA chaperone proteins that function by disrupting structure, which can facilitate structural conversions. Here, studies with both local and global focuses are used to uncover mechanistic features of DEAD-box proteins CYT-19 and Mss116p. Both of these proteins are general RNA chaperones as they each have the ability to facilitate proper folding of multiple structured RNAs. The first study probes how DEAD-box proteins interact with a simple duplex substrate. Separating the strands of a duplex is an ATP-dependent process and is central to structural disruption by DEAD-box proteins. Here, how ATP is utilized during duplex separation is monitored by comparing ATP hydrolysis rates with strand separation rates. Results indicate that one ATP molecule is sufficient for complete separation of a 6-11 base pair RNA duplex. Under some conditions, ATP binding in the absence of hydrolysis is sufficient for duplex separation. Next, focus is shifted to a more global perspective as the function of Mss116p is probed in the folding of a cognate group II intron substrate, aI5[gamma], under near-physiological conditions. Three catalytically-active constructs of aI5[gamma] are used and catalysis serves as a proxy for folding. Folding of all constructs is promoted by the presence of Mss116p and ATP. In vitro and in vivo results indicate that a local unfolding event is promoted by Mss116p, stimulating formation of the native state. Lastly, orthogonal methods that probe physical features of RNA are used to provide insight into the structural intermediates with which Mss116p acts.