DEAD-box proteins can completely separate an RNA duplex using a single ATP.
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.
Chen
Yingfeng
Y
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Potratz
Jeffrey P
JP
Tijerina
Pilar
P
Del Campo
Mark
M
Lambowitz
Alan M
AM
Russell
Rick
R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000502:physiology*; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012330:RNA, Double-Stranded / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*
D012330:RNA, Double-Stranded; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C492152:DED1 protein, S cerevisiae; C058150:MSS116 protein, S cerevisiae; D053487:DEAD-box RNA Helicases