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Structural Inference of Native and Partially Folded RNA by High-Throughput Contact Mapping

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Structural Inference of Native and Partially Folded RNA by High-Throughput Contact Mapping Structural inference of native and partially folded RNA by high-throughput contact mapping Rhiju Das*†, Madhuri Kudaravalli*, Magdalena Jonikas‡, Alain Laederach§, Robert Fong¶, Jason P. Schwans*, David Bakerʈ, Joseph A. Piccirilli¶, Russ B. Altman‡§, and Daniel Herschlag*,** Departments of *Biochemistry, ‡Bioengineering, and §Genetics, Stanford University, Stanford, CA 94305; ¶Department of Chemistry, University of Chicago, Chicago, IL 60637; and ʈDepartment of Biochemistry, University of Washington, Seattle, WA 98195 Edited by Alan M. Lambowitz, University of Texas, Austin, TX, and approved December 18, 2007 (received for review September 24, 2007) The biological behaviors of ribozymes, riboswitches, and numer- and RNA/protein complexes function through structures in which ous other functional RNA molecules are critically dependent on only a portion of the RNA molecule adopts a discrete three- their tertiary folding and their ability to sample multiple functional dimensional fold (4, 8, 9). Overall, understanding the fundamental states. The conformational heterogeneity and partially folded properties, folding pathways, and energetics of functional RNA nature of most of these states has rendered their characterization molecules requires information about intermediates in the folding by high-resolution structural approaches difficult or even intrac- process and not just a single final folded state (10). The partially table. Here we introduce a method to rapidly infer the tertiary folded nature of these intermediates renders their characterization helical arrangements of large RNA molecules in their native and by x-ray crystallography and NMR particularly difficult and possibly non-native solution states. Multiplexed hydroxyl radical (⅐OH) intractable; structural information for these states, even at a modest cleavage analysis (MOHCA) enables the high-throughput detection spatial resolution, would be a boon for the understanding of RNA of numerous pairs of contacting residues via random incorporation behavior. of radical cleavage agents followed by two-dimensional gel elec- The difficulty of inferring RNA structure is underscored by trophoresis. We validated this technology by recapitulating the continuing investigations of the first large globular RNA whose unfolded and native states of a well studied model RNA, the P4–P6 native structure was determined by crystallography (11, 12), the domain of the Tetrahymena ribozyme, at subhelical resolution. We 158-nucleotide P4–P6 domain of the Tetrahymena thermophila then applied MOHCA to a recently discovered third state of the group I intron. Despite its status as one of the best-studied model P4–P6 RNA that is stabilized by high concentrations of monovalent systems in RNA folding (see, e.g., refs. 13–17), the structure of a salt and whose partial order precludes conventional techniques for partially folded state of this RNA, discovered in studies at high structure determination. The three-dimensional portrait of a com- monovalent ion concentrations (17), remains unknown. This state, pact, non-native RNA state reveals a well ordered subset of native attained under unusual solution conditions, mimics a kinetic and tertiary contacts, in contrast to the dynamic but otherwise similar thermodynamic folding intermediate observed in folding pathway molten globule states of proteins. With its applicability to nearly of the P4–P6 RNA and its mutants under physiological conditions any solution state, we expect MOHCA to be a powerful tool for (17). As illustrated by the insightful studies of molten globule states illuminating the many functional structures of large RNA molecules in protein folding (see, e.g., ref. 18), a complete picture of RNA and RNA/protein complexes. structure and energetics will be greatly aided by understanding such folding intermediates and their analogs. hydroxyl radical ͉ molten globule ͉ Tetrahymena ribozyme ͉ We present a high-throughput contact-mapping technique that two-dimensional gel permits the rapid inference of the helical arrangement of RNA conformations and apply it to three states of the P4–P6 RNA: the unfolded state, the folded state, and the hitherto unsolved partially he discoveries of catalytic RNAs, silencing RNAs, riboswitches, folded state. Although currently at modest resolution, these struc- and a panoply of functional RNA molecules have sweeping T tural portraits clearly illustrate physical distinctions between the implications for our views of evolution from an early ‘‘RNA World’’ three states and illuminate informative similarities and differences and for the potential of structured RNAs to act in roles beyond the relative to protein folding intermediates. High-throughput contact simple transmission of information laid out in the Central Dogma mapping holds immediate promise for revealing previously unavail- of Molecular Biology (1). The functions of these RNAs in primitive able structural information for the many folded and partially folded and modern life are being elucidated at an explosive pace. Never- states of large RNAs. theless, a deep understanding of these fundamental biopolymers and their biological roles requires structural portraits of their Results functional states, and, in this respect, progress has been slow. Contact Mapping by Multiplexed Hydroxyl Radical Cleavage Analysis. Our understanding of RNA structure has greatly lagged behind Without crystallographic diffraction data, pairwise distance con- that of protein structure: compared with nearly 40,000 protein straints are the most valuable type of data for structural inference, structures in the Protein Data Bank, there are currently Ͻ1,000 experimentally determined RNA structures, most of which are small fragments (2). High-resolution approaches using NMR spec- Author contributions: R.D., A.L., D.B., R.B.A., and D.H. designed research; R.D., M.K., and troscopy (NMR) and x-ray crystallography have the potential to M.J. performed research; R.D., R.F., J.P.S., and J.A.P. contributed new reagents/analytic describe RNA structure at the atomic level, but have been consid- tools; R.D. analyzed data; and R.D. and D.H. wrote the paper. erably hampered by numerous factors, including limited chemical The authors declare no conflict of interest. shift dispersion, the large sizes of structured RNAs, and the poor This article is a PNAS Direct Submission. behavior of RNA at high concentrations. Freely available online through the PNAS open access option. Further enriching and complicating the modeling of RNA be- †Present address: Department of Biochemistry, University of Washington, Seattle, WA havior is the seemingly pervasive tendency of RNA to form 98195. alternative secondary and tertiary structures. Indeed, cycling be- **To whom correspondence should be addressed. E-mail: [email protected]. tween partially structured folding intermediates or alternative This article contains supporting information online at www.pnas.org/cgi/content/full/ conformations appears to be crucial to the versatility and function 0709032105/DC1. of RNAs in their cellular milieu (3–7). Furthermore, many RNAs © 2008 by The National Academy of Sciences of the USA 4144–4149 ͉ PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709032105 5´ cleavage agent and the residues cleaved by that particular agent. A O 5´ 3´ O P O- O O O Combining the resulting data with new computational tools then O - O C S- O P O P O P O O A Step 1 Step 2 O- O- NH2 S- O OH generates three-dimensional structures of folding states. The tech- OH O P S- γ−32 NH2 P ATP T7 polymerase O A O S- ⅐ ATP, GTP, T4 Kinase nique, which we term Multiplexed OH Cleavage Analysis (MO- O NH 32 NH UTP, CTP - 2 O P O P HCA), is briefly summarized below and in Fig. 1A. A complete DNA Template O O A O OH step-by-step protocol is given in the supporting information (SI) 5´ 3´ O Text. O P S- 5´ O O A Step 5 Steps 6 & 7 Incorporation of the cleavage agent into a large RNA is accom- Steps 3 & 4 HN S O O - OO S- - - O P O HN O -O 2+ plished by in vitro transcription of the RNA in the presence of a O O A N O Gel Fe Mg •OH S- ITCB-EDTA O- Fe O purify & Ascorbate Fe Fe(III) O OH N •OH O P O- elute S- •OH •OH modified nucleotide triphosphate (step 1, Fig. 1A). The nucleotide O cleavage 3´ ITCB-EDTA Fe 3´ contains a 2Ј-amino modification, which allows attachment of the Position of •OH Cleavage Agent hydroxyl radical cleavage agent, and a phosphorothioate group, Position of •OH Cleavage Position of •OH Cleavage Step 8 Step 9 Step 10 which allows specific cleavage at a later stage (24); this ‘‘tagging’’ strategy has been widely used for high-throughput mutational Gel I 2 Gel sorting Cleavage sorting analysis (25). After random incorporation of the modified nucle- by at by size: phosphoro- size: st otide at a frequency of approximately one modification per RNA 1 thioate 2nd e direction direction (step 1), the RNA is end-radiolabeled (step 2) and the cleavage agent, an Fe-EDTA chelate, is tethered to the 2Ј-amino groups that B are present throughout the RNA molecules (steps 3 and 4). This A115 C D A123 A133 A146 A140 A pool of RNA molecules is purified (step 5) and folded to the desired C A G A C A G C U A G C 200 G C state (step 6), after which localized radical generation is initiated by P5a C GU G U 120 A U U G G C P5 C G U A C G C G reduction of Fe(III) to Fe(II) and then quenched after several G G A U U A A U A A A A minutes (step 7). The cleavage products are first separated by length U A A A 180 G C G C C G P5c UA C G G U A A 260 A A AGG C G via polyacrylamide gel electrophoresis (step 8). This pattern indi- C G C CA P4 C G U G U C C U G G A 140 A U U G cates where each RNA was cleaved but not the location of the A U G U U 160 G C P6 C G A A G G A U G C C A responsible cleavage agent.
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