DEAD-box proteins can completely separate an RNA duplex using a single ATP

Abstract

DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6 -11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably <1 ATP is hydrolyzed per separation event, even though strand separation is strongly dependent on ATP and is not supported by the nucleotide analog AMP-PNP. Thus, ATP strongly enhances strand separation activity even without being hydrolyzed, most likely by eliciting or stabilizing a protein conformation that promotes strand separation, and AMP-PNP does not mimic ATP in this regard. Together, our results show that DEADbox proteins can disrupt short duplexes by using a single cycle of ATP-dependent conformational changes, strongly supporting and extending models in which DEAD-box proteins perform local rearrangements while remaining tethered to their target RNAs or RNP complexes. This mechanism may underlie the functions of DEAD-box proteins by allowing them to generate local rearrangements without disrupting the global structures of their targets.CYT-19 ͉ group I intron ͉ RNA chaperone ͉ RNA folding ͉ RNA helicase S tructured RNAs and RNA-protein complexes (RNPs) mediate a host of essential cellular processes, including processing of messenger RNAs and their translation into protein. In addition to folding into defined structures, many of these RNAs and RNPs undergo extensive conformational changes during their functions. Both their initial folding and conformational changes typically require DEAD-box proteins, which use ATP to promote RNA structural transitions.DEAD-box proteins are members of helicase superfamily-2 (SF2) and are related to the ATP-dependent RNA and DNA helicases that function in replication and other aspects of nucleic acid metabolism (1). However, rather than unwinding long, continuous duplexes, many DEAD-box proteins manipulate highly structured RNAs and RNPs by facilitating rearrangements that can include local disruptions of secondary structure, tertiary structure, and RNA-protein interactions.Consistent with their distinct functions, recent in vitro studies have strongly suggested that DEAD-box proteins operate on structured RNAs by a mechanism that is fundamentally different from processive helicases. The Neurospora crassa CYT-19 protein is required for proper folding of several mitochondrial group I introns in Neurospora crassa (2). It also assists folding of diverse group I and group II introns in vitro or when expressed in...

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…Duplex unwinding can be achieved by hydrolysis of one ATP molecule, and under certain conditions less than one ATP is hydrolyzed per duplex separation event (Chen et al, 2008). In fact, ATP bound to the helicase core was only hydrolyzed in half of the strand separation events (Chen et al, 2008), further supporting that strand separation can precede hydrolysis. Strikingly, ADP-BeF x does not support unwinding by the DExH box protein NPH-II (Liu et al, 2008).…”

“…DEAD box protein mediated duplex unwinding requires ATP, and it has been inferred that the energy from ATP hydrolysis is required for base pair disruption. By contrast, many DEAD box proteins already promote limited strand separation in the absence of ATP, which has been interpreted as nonspecific duplex destabilization via an ssRNA capture mechanism Chen et al, 2008). For a few DEAD box proteins, a strand annealing activity has been demonstrated (Yang and Jankowsky, 2005;Halls et al, 2007).…”

“…In contrast, ADP-AlF x and ADP-P i do not support RNA unwinding, suggesting that strand separation occurs prior to ATP hydrolysis (Liu et al, 2008). Duplex unwinding can be achieved by hydrolysis of one ATP molecule, and under certain conditions less than one ATP is hydrolyzed per duplex separation event (Chen et al, 2008). In fact, ATP bound to the helicase core was only hydrolyzed in half of the strand separation events (Chen et al, 2008), further supporting that strand separation can precede hydrolysis.…”

“…Consequently, ATP hydrolysis must be required to reset the enzyme and to enable multiple turnovers (Liu et al, 2008). ADP-BeF x appears to faithfully mimic the ATP conformation of DEAD box proteins, whereas it has been suggested that ADPNP traps a non-productive DEAD box protein/RNA complex (Henn et al, 2008) and does not elicit the same conformation of DEAD box proteins as ATP (Chen et al, 2008;Liu et al, 2008). Although ADPNP does not support RNA unwinding, its binding to the helicase core allows for the closure of the interdomain cleft in response to RNA binding (Andersen et al, 2006;Bono et al, 2006;Theissen et al, 2008;Karow and Klostermeier, 2009;von Moeller et al, 2009).…”

“…Product release is coupled to the dissociation of the second RNA strand (step 5), thereby resetting the enzyme for further cycles. Possibly, ATP can dissociate before being hydrolyzed (gray, right arm), allowing for the completion of one cycle of unwinding in the absence of ATP hydrolysis (Chen et al, 2008). For longer RNA duplexes, destabilization by the kink does not lead to the same probability for complete strand separation, and dissociation of the first RNA strand from the activated complex.…”