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

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DEAD-Box Proteins Can Completely Separate an RNA Duplex Using a Single ATP DEAD-box proteins can completely separate an RNA duplex using a single ATP Yingfeng Chen1, Jeffrey P. Potratz1, Pilar Tijerina, Mark Del Campo, Alan M. Lambowitz, and Rick Russell2 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 (sent for review July 10, 2008) DEAD-box proteins are ubiquitous in RNA metabolism and use ATP intron junction, and that this unwinding efficiency (kcat/KM)is to mediate RNA conformational changes. These proteins have been enhanced by 2 orders of magnitude when the duplex is covalently suggested to use a fundamentally different mechanism from the linked to the ribozyme compared with the same duplex in related DNA and RNA helicases, generating local strand separation solution (4). Then, using simple constructs based on group I while remaining tethered through additional interactions with intron structure, we found that the activity was also enhanced by structured RNAs and RNA-protein (RNP) complexes. Here, we simple extensions to the helix. Interestingly, a single-stranded provide a critical test of this model by measuring the number of extension gave a smaller enhancement than a double-stranded ATP molecules hydrolyzed by DEAD-box proteins as they separate flanking region, and both gave smaller enhancements than the short RNA helices characteristic of structured RNAs (6–11 bp). We highly structured intact group I intron. show that the DEAD-box protein CYT-19 can achieve complete Whereas conventional DNA and RNA helicases commonly strand separation using a single ATP, and that 2 related proteins, require a 5Ј-or3Ј-single-stranded region, which serves as a Mss116p and Ded1p, display similar behavior. Under some condi- starting point for translocation into and through the duplex, tions, considerably <1 ATP is hydrolyzed per separation event, results above suggested that the increased activity arose instead even though strand separation is strongly dependent on ATP and from an additional and distinct interaction of CYT-19 with the is not supported by the nucleotide analog AMP-PNP. Thus, ATP RNA. Further, the enhancement under subsaturating conditions strongly enhances strand separation activity even without being (kcat/KM) suggested that this interaction is maintained in the hydrolyzed, most likely by eliciting or stabilizing a protein confor- transition state for strand separation. Additional work showed mation that promotes strand separation, and AMP-PNP does not that the enhancement is nearly eliminated by deletion of 49 aa mimic ATP in this regard. Together, our results show that DEAD- from the highly basic C terminus of CYT-19, whereas strand box proteins can disrupt short duplexes by using a single cycle of separation of a duplex that lacks an extension is essentially ATP-dependent conformational changes, strongly supporting and unaffected, most simply suggesting that this additional interac- extending models in which DEAD-box proteins perform local re- tion is mediated by the C-terminal region (6). arrangements while remaining tethered to their target RNAs or Together, these findings led to a model in which interactions RNP complexes. This mechanism may underlie the functions of with adjacent RNA structure tether DEAD-box proteins in DEAD-box proteins by allowing them to generate local rearrange- proximity to exposed helices or perhaps other elements of RNA ments without disrupting the global structures of their targets. structure, where binding of the core domain and ATP- dependent conformational changes give strand separation (4, 7, CYT-19 ͉ group I intron ͉ RNA chaperone ͉ RNA folding ͉ RNA helicase 8). Although early studies demonstrating that DEAD-box pro- teins can readily separate duplexes of Ϸ1 helical turn or less but are essentially inactive for duplexes of 2 or more turns (9, 10) tructured RNAs and RNA-protein complexes (RNPs) me- indicated a lack of processivity, the tethering model suggests a diate a host of essential cellular processes, including pro- S more radical difference in mechanism from processive helicases. cessing of messenger RNAs and their translation into protein. In This is because continuous formation of a tethering interaction addition to folding into defined structures, many of these RNAs during duplex unwinding would most simply suggest the absence and RNPs undergo extensive conformational changes during of any translocation during the unwinding process. their functions. Both their initial folding and conformational Strong independent support for essential features of this changes typically require DEAD-box proteins, which use ATP to model has come from studies in the Jankowsky laboratory, using promote RNA structural transitions. an elegant set of model duplex substrates. First, they demon- DEAD-box proteins are members of helicase superfamily-2 strated conclusively that a flanking sequence can enhance ac- (SF2) and are related to the ATP-dependent RNA and DNA tivity for DEAD-box proteins without serving as a starting point helicases that function in replication and other aspects of nucleic for translocation by showing that a single-stranded segment can acid metabolism (1). However, rather than unwinding long, still give activation if it is not linked to the target duplex but is continuous duplexes, many DEAD-box proteins manipulate instead bound through biotin-mediated interactions with the highly structured RNAs and RNPs by facilitating rearrange- protein streptavidin (7). Second, they showed that even model ments that can include local disruptions of secondary structure, duplexes with an RNA segment flanked on both sides by DNA tertiary structure, and RNA-protein interactions. can be efficiently separated by DEAD-box proteins, whereas the Consistent with their distinct functions, recent in vitro studies same proteins are not active on fully DNA substrates, indicating 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 pro- Author contributions: Y.C., J.P.P., P.T., and R.R. designed research; Y.C., J.P.P., P.T., and tein is required for proper folding of several mitochondrial group M.D.C. performed research; Y.C., J.P.P., P.T., M.D.C., A.M.L., and R.R. analyzed data; and I introns in Neurospora crassa (2). It also assists folding of diverse Y.C., A.M.L., and R.R. wrote the paper. group I and group II introns in vitro or when expressed in yeast The authors declare no conflict of interest. (3–5), indicating that it acts as a general RNA chaperone. 1Y.C. and J.P.P. contributed equally to this work To gain insight into the mechanism of its activity, we took 2To whom correspondence should be addressed. E-mail: rick࿝[email protected]. advantage of the observation that CYT-19 can use its nonspecific This article contains supporting information online at www.pnas.org/cgi/content/full/ chaperone activity to efficiently separate the 6-base pair helix 0811075106/DCSupplemental. BIOCHEMISTRY termed P1, formed between group I introns and their 5Ј-exon- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811075106 PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20203–20208 Downloaded by guest on September 23, 2021 that strand separation can be initiated internally, without trans- A Activating P1 location (11). The main features of the model are also indicated DNA duplex RNA duplex AC 5' for the Escherichia coli DEAD-box protein DbpA by studies T GGACTATTGAAAAGGGAGG G • • • 3' from Uhlenbeck and colleagues, with the important difference C ACCTGGTAGCTA CCCUCUAAAAA that this protein uses an ancillary domain to recognize a par- ticular structure within the large subunit ribosomal RNA rather 8 B C 0.6 than interacting with structured RNA more generally (12–15). 6 In the current work, we have tested and extended this model •• • 0.4 – CYT-19 by measuring the number of ATP molecules used by CYT-19 and 4 Pi (µM) 0.2 other DEAD-box proteins as they separate RNA duplexes. In the 2 •• Fraction duplex + CYT-19 most extreme form of the model, with strand separation accom- 0 51015 0 10 20 30 plished in the presence of a continuously formed tethering Time (min) Time (min) interaction, it would be possible that the complete reaction Duplex-dependent ATP hydrolysis CYT-19-dependent strand separation would be accomplished in a single cycle of ATP-dependent conformational changes and would therefore give hydrolysis of •• • + CYT-19 – CYT-19 only 1 ATP. Indeed, we obtain this result for duplexes of 6–11 0.80 µM•min–1 – 0.06 µM•min–1 = (1.5 min–1 – 0.08 min–1) X 0.5 µM duplex = bp, characteristic of helices present in structured RNAs. Further, 0.74 µM•min–1 0.69 µM•min–1 under some conditions, a significant fraction of strand- separation events occur in the absence of any ATP hydrolysis. Fig. 1. ATP hydrolysis and RNA strand separation by CYT-19. (A) Structure of the duplex substrate, derived from the P1 duplex of the Tetrahymena group Nevertheless, these events are dependent on ATP, indicating I intron. RNA nucleotides are red and DNA nucleotides are black. The DNA that bound ATP favors a protein conformation that promotes portion has the equivalent sequence of the P2 helix, which is adjacent to P1 in local strand separation even before ATP hydrolysis. the natural intron. (B) P1 duplex-dependent ATP hydrolysis by CYT-19. ATP hydrolysis was measured in the presence of the P1 duplex by including both Results oligonucleotides (F), or with the same concentrations of the RNA/DNA oligo- ␮ E ␮ In designing a duplex substrate for these studies, we took nucleotide (0.5 M, ) or the excess CCCUCUA5 (1 M, triangles) alone. (C) advantage of our previous results that CYT-19 can efficiently Strand separation of the P1 duplex construct in the presence (filled circles) or ␮ separate the P1 duplex of the Tetrahymena group I ribozyme, absence (open circles) of CYT-19.
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