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

doi:10.1073/pnas.0811075106

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

Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712

Contributed by Alan M. Lambowitz, November 4, 2008
↵¹Y.C. and J.P.P. contributed equally to this work (received for review July 10, 2008)

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 DEAD-box 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.

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Footnotes

²To whom correspondence should be addressed. E-mail: rick_russell{at}mail.utexas.edu
Author contributions: Y.C., J.P.P., P.T., and R.R. designed research; Y.C., J.P.P., P.T., and M.D.C. performed research; Y.C., J.P.P., P.T., M.D.C., A.M.L., and R.R. analyzed data; and Y.C., A.M.L., and R.R. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0811075106/DCSupplemental.
↵* The CYT-19 concentration can be varied only over a limited range with our current methods because at low concentrations the ATPase rate becomes too small to measure the fraction of ATP hydrolyzed, and at high CYT-19 concentrations, the strand separation becomes too fast for hand pipetting. Across the accessible range (0.5–2 μM, with CYT-19 in 4-fold excess of the duplex), the rate constant for strand separation increased approximately linearly, indicating that CYT-19 is subsaturating with respect to the duplex, and the ATP utilization value was unchanged within the expected limits of uncertainty (data not shown).
↵† This value arises from the strand separation rate in the presence of ATP under conditions that do not favor its hydrolysis. The rate is 2–4 μM/min with 150 μM ATP, with no indication of saturation (data not shown), and therefore at least 8 μM/min with saturating ATP. ATP is hydorlyzed in only half of the strand-separation events. Thus, the pathway that involves bound ATP but not its hydrolysis must give half of the total rate (4 μM/min), ≥20-fold faster than CYT-19-dependent strand separation in the absence of ATP.