We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (< or = 500 nt). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as 'control' conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a 'footprint'), it is possible to detect local changes in the secondary and tertiary structure of RNA, as well as the formation of RNA-protein contacts. This protocol takes 1.5-3 d to complete, depending on the type of analysis used.

We explore the interactions of CYT-19, a DExD͞H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD͞H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.group I RNA ͉ RNA folding ͉ RNA unwinding ͉ Tetrahymena ribozyme E ssentially all cellular processes that are mediated by structured RNAs also require one or more DExD͞H-box proteins (1). These proteins use the energy from ATP binding and hydrolysis to accelerate RNA structural transitions, which can represent folding steps toward the native state or conformational switches between functional forms. The requirement for proteins presumably arises because RNA base pairs and other local structure can be highly stable even in the absence of enforcing structure, such that folding steps or rearrangements that require significant unfolding require assistance to proceed efficiently (2-4).Despite their ubiquitous presence, key questions about the functions of DExD͞H-box proteins remain largely unanswered. First, what interactions direct different DExD͞H-box proteins to their physiological substrates? All of these proteins share a core ''helicase'' domain containing a set of conserved motifs, and most have additional domains, a few of which have been shown to recognize substrate RNAs or RNA-protein complexes (reviewed in ref. 5). On this basis, targeting has been proposed as a general role for these domains, and the specific interactions that target one DExD͞H-box protein have been delineated (6-9). Nevertheless, in general, the interactions that direct DExD͞H box proteins to their substrates remain to be identified.Second, h...

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 DEAD-box protein CYT-19 functions in folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA binding site, distinct from the unwinding active site, which enhances unwinding activity by binding non-specifically to adjacent RNA structure. Here we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or to single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, as the truncated protein is less active in facilitating folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs so that it can efficiently disrupt exposed, non-native structural elements, allowing them to re-fold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of them may function similarly as non-specific RNA-binding elements that enhance general RNA chaperone activity.Structured RNAs are required for a myriad of cellular processes, including mRNA processing and translation, tRNA processing, and maintenance of chromosome ends, and nearly all structured RNAs require at least one DExD/H-box protein for their functions (2,3). DExD/Hbox proteins are thought to function in large part by facilitating structural transitions of RNAs and ribonucleoprotein (RNP) complexes that would otherwise be too slow to allow the complexes to form or function. DExD/H-box proteins include a conserved motor domain, which uses energy derived from cycles of ATP binding and hydrolysis to facilitate structural rearrangements of RNAs, at least in part by 'unwinding' double-stranded segments (2,4). Many † This work was supported by grants from the NIH (R01-GM070456 to R.R. and R01-GM037951 to A.M.L.) and from the Welch Foundation (F-1563 to R.R.). M.D. was supported by a postdoctoral fellowship from the NIH (F01-GM076961).*To whom correspondence should be addressed. Tel: 512-471-1514; Fax: 512-232-3432; E-mail: rick_russell@mail.utexas.edu. 1 Abbreviations: Δ578-626, C-terminal truncation variant of CY...

The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.