Local RNA structural changes induced by crystallization are revealed by SHAPE

Vicens, Quentin; Gooding, Anne R.; Laederach, Alain; Cech, Thomas R.

doi:10.1261/rna.400207

Cited by 52 publications

(60 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…They may reflect alternative structures induced by a small subset of incorporated cleavage sources or may signal intrinsic RNA solution dynamics not captured in the reference crystal structure. The latter effects have been suggested by chemical cross-linking measurements (31,32) and are supported by localized discrepancies of footprinting data for the RNA in solution with footprinting data for the RNA in crystallized form (33) and with the solvent-accessible surface area computed for the P4-P6 crystal structure (SI Fig. 13).…”

Section: Internal Consistency and Accuracy Of High-throughput Contactmentioning

confidence: 65%

Structural inference of native and partially folded RNA by high-throughput contact mapping

Das¹,

Kudaravalli²,

Jonikas

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The biological behaviors of ribozymes, riboswitches, and numerous other functional RNA molecules are critically dependent on their tertiary folding and their ability to sample multiple functional states. The conformational heterogeneity and partially folded nature of most of these states has rendered their characterization by high-resolution structural approaches difficult or even intractable. Here we introduce a method to rapidly infer the tertiary helical arrangements of large RNA molecules in their native and non-native solution states. Multiplexed hydroxyl radical (⅐OH) cleavage analysis (MOHCA) enables the high-throughput detection of numerous pairs of contacting residues via random incorporation of radical cleavage agents followed by two-dimensional gel electrophoresis. We validated this technology by recapitulating the unfolded and native states of a well studied model RNA, the P4 -P6 domain of the Tetrahymena ribozyme, at subhelical resolution. We then applied MOHCA to a recently discovered third state of the P4 -P6 RNA that is stabilized by high concentrations of monovalent salt and whose partial order precludes conventional techniques for structure determination. The three-dimensional portrait of a compact, non-native RNA state reveals a well ordered subset of native tertiary contacts, in contrast to the dynamic but otherwise similar molten globule states of proteins. With its applicability to nearly any solution state, we expect MOHCA to be a powerful tool for illuminating the many functional structures of large RNA molecules and RNA/protein complexes.hydroxyl radical ͉ molten globule ͉ Tetrahymena ribozyme ͉ two-dimensional gel T he discoveries of catalytic RNAs, silencing RNAs, riboswitches, and a panoply of functional RNA molecules have sweeping implications for our views of evolution from an early ''RNA World'' and for the potential of structured RNAs to act in roles beyond the simple transmission of information laid out in the Central Dogma of Molecular Biology (1). The functions of these RNAs in primitive and modern life are being elucidated at an explosive pace. Nevertheless, a deep understanding of these fundamental biopolymers and their biological roles requires structural portraits of their functional states, and, in this respect, progress has been slow.Our understanding of RNA structure has greatly lagged behind that of protein structure: compared with nearly 40,000 protein 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 spectroscopy (NMR) and x-ray crystallography have the potential to describe RNA structure at the atomic level, but have been considerably hampered by numerous factors, including limited chemical shift dispersion, the large sizes of structured RNAs, and the poor behavior of RNA at high concentrations.Further enriching and complicating the modeling of RNA behavior is the seemingly pervasive tendency of RNA to form alternative secondary and tert...

show abstract

Section: Internal Consistency and Accuracy Of High-throughput Contactmentioning

confidence: 65%

Structural inference of native and partially folded RNA by high-throughput contact mapping

Das¹,

Kudaravalli²,

Jonikas

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…In support of this, X-ray crystallography of an octanucleotide RNA/DNA hybrid derived from the HIV-1 PPT has shown that anomalous stacking renders the À12a/À11g/À10a step highly deformable, which may be sufficient to promote transient loss of base-pairing (Kopka et al 2003). Alternatively, À12a/À11g NMIA sensitivity may reflect an alteration in sugar pucker from the C39-endo to C29-endo conformation, which has been suggested to mediate ligand recognition (which in this case would be HIV RT) and catalysis (Vicens et al 2007;Gherghe et al 2008).…”

Section: Nmia Sensitivity Of Hiv-1 Ppt Ribonucleotidesmentioning

confidence: 99%

“…Primer extension subsequently creates a cDNA library corresponding to termination at sites of adduct formation. While SHAPE has elucidated two-dimensional structures of several regulatory RNAs (Badorrek and Weeks 2006;Wilkinson et al 2006;Vicens et al 2007;Gherghe et al 2008;Wang et al 2008;Wilkinson et al 2008), there are no reports on its use to study the architecture of RNA in the context of RNA/DNA hybrids. Moreover, the size and purine-rich sequence of wild-type and analog-substituted RNA/DNA hybrids (20-25 base pairs [bp]) made the primer extension step impractical.…”

Section: Introductionmentioning

confidence: 99%

SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Turner¹,

Yi-Brunozzi²,

Brinson³

et al. 2009

RNA

View full text Add to dashboard Cite

Selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 29-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)-containing RNA/DNA hybrids, their size (20-25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M r = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 29-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.

show abstract

“…A similar distribution of reactive and unreactive C2′-endo nucleotides occurs in the Tetrahymena P5-P4-P6 domain using NMIA. 8 While the C2′-endo conformation by itself clearly does not govern SHAPE reactivity, a distinct class of C2′-endo nucleotides experiences extraordinarily slow local dynamics. These nucleotides in both the simple C2′-endo RNA ( Figure 2B) and the RNase P RNA ( Figure 4B) share key characteristics: (1) the ribose group has the C2′-endo conformation, and (2) the nucleotide conformation is partially constrained by base stacking and hydrogen bonding interactions ( Figure S4).…”

mentioning

confidence: 99%

Slow Conformational Dynamics at C2′-endo Nucleotides in RNA

et al. 2008

View full text Add to dashboard Cite

Local and global dynamics in folded RNAs occur over broad timescales spanning picoseconds to minutes. 1 Slow motions likely play predominant roles in governing RNA folding and ribonucleoprotein assembly reactions. However, slow local motions are extremely difficult to detect, especially for large RNAs with complex structures.The local environment and degree of flexibility can be evaluated at nucleotide resolution for RNAs of any size using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry. 2 RNA nucleotides exist in equilibrium between constrained (closed) and flexible (open) states. The 2′-OH group in flexible nucleotides preferentially adopts an open, reactive, conformation that facilitates reaction with electrophilic reagents to form a 2′-O-adduct (Figure 1). SHAPE experiments work well using electrophiles based on the isatoic anhydride (IA) scaffold. 2a,3 Positions that form 2′-O-adducts are detected by primer extension. 2 IA derivatives both react with the RNA 2′-OH group and also undergo concurrent degradation by hydrolysis (Figure 1). 2′-OH reactivity is thus conveniently monitored by allowing a reaction to proceed until the reagent has been consumed, either by hydrolysis or reaction with RNA. At this end point, the fraction adduct at any nucleotide (f) is (1) Where (2) and the rate of hydrolysis has been shown to be proportional to the rate of adduct formation, 2b,3 k adduct /k hydrolysis = β. These relationships lead to two limits. In limit 1, k open + k close ≫ k adduct [reagent] Correspondence to: Kevin M. Weeks, weeks@unc.edu. Supporting Information Available: Methods and four figures. This material is available free of charge via the Internet at http:// pubs.acs.org. HHS Public AccessIt should therefore be possible to monitor local nucleotide dynamics in RNA under conditions where limit 2 applies by varying the reactivity (or k hydrolysis ) of the hydroxylselective electrophile. IA has a hydrolysis half-life (t 1/2 ) of 430 s at 37 °C. Electronwithdrawing substituents at the cyclic amine (R 1 ) or in the benzene ring (R 2 ) enhance reagent reactivity. Compared to IA, N-methyl isatoic anhydride (NMIA), 4-nitroisatioc anhydride (4NIA), and 1-methyl 7-nitroisatoic anhydride (1M7) 3 have progressively shorter hydrolysis half-lives (table, Figure 1).To investigate if distinct local nucleotide dynamics can be captured by varying the SHAPE electrophile, we focused on an important variation in RNA structure: the C2′-endo conformation. Although C2′-endo nucleotides are relatively rare, they are highly overrepresented in important RNA tertiary interactions and in catalytic active sites. 4 Local structure at tandem G•A mismatches depends on the local sequence context. 5 Guanosine nucleotides in G•A pairs adopt the C2′-endo conformation in the sequences (UGAA) 2 5a and (GGAU) 2 , 5b the C3′-endo conformation typical of standard A-form helix geometry in (CGAG) 2 , 5c and a mixture of C2′-endo/C3′-endo conformations in (UGAG) 2 . 5b We constructed a simple hairpin RNA (termed...

show abstract

Local RNA structural changes induced by crystallization are revealed by SHAPE

Cited by 52 publications

References 52 publications

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

SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Slow Conformational Dynamics at C2′-endo Nucleotides in RNA

Contact Info

Product

Resources

About