DEAD-Box Proteins Can Completely Separate an RNA Duplex Using a Single ATP

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 51; pp. 20203 - 20208
• Main Authors: Chen, Yingfeng, Potratz, Jeffrey P., Tijerina, Pilar, Campo, Mark Del, Lambowitz, Alan M., Russell, Rick
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 23.12.2008; National Acad Sciences
• Subjects: Adenosine triphosphatase; Adenosine triphosphatases; Adenosine Triphosphate - metabolism; Biochemistry; Biological Sciences; Cell cycle; DEAD-box RNA Helicases - chemistry; DEAD-box RNA Helicases - physiology; DNA; Hydrolysis; Introns; Models, Chemical; Molecules; Nucleic Acid Conformation; Nucleotides; Oligonucleotides; Protein Conformation; Proteins; Ribonucleic acid; RNA; RNA, Double-Stranded - metabolism; Saccharomyces cerevisiae Proteins - physiology; Splicing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0811075106

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.