Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

Bailor, Maximillian H.; Musselman, Catherine A.; Hansen, Alexandar L.; Gulati, Kush; Patel, Dinshaw J.; Al‐Hashimi, Hashim M.

doi:10.1038/nprot.2007.221

Cited by 57 publications

(78 citation statements)

References 123 publications

(154 reference statements)

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“…Although powerful, these NMR-based methods, like all approaches, have limitations. For example, RDCs have difficulty distinguishing between conformations with similar angular orientations but different translational displacements (15,16). Additional methods are needed to construct ensembles that can test and complement these current methods.…”

Section: Helix-junction-helix | Saxsmentioning

confidence: 99%

From a structural average to the conformational ensemble of a DNA bulge

Shi¹,

Beauchamp

Harbury

et al. 2014

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

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Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurinesubstituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods. helix-junction-helix | SAXSA grand challenge in biology is to understand the complex free-energy landscape of macromolecules and to decipher the resulting conformational ensembles. To perform their biological functions, macromolecules must adopt a multiplicity of conformations. Balancing and controlling different conformational states is central to biological processes including protein folding, allostery and signaling, and the stepwise assembly and function of macromolecular machines. To understand these complex molecules requires characterization of their free-energy landscapes-i.e., their equilibrium conformational ensembles. Precise measurements of conformational ensembles could allow quantitative modeling of the folding and function of biological macromolecules, would provide valuable experimental data to test current computational models and assumptions, and might facilitate the rational design of specifically acting inhibitors (1, 2).Techniques including NMR and EPR relaxation have been developed to incisively probe motions in the ensemble on different time scales, ranging from pic...

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Section: Helix-junction-helix | Saxsmentioning

confidence: 99%

From a structural average to the conformational ensemble of a DNA bulge

Shi¹,

Beauchamp

Harbury

et al. 2014

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

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“…To gain further insights into the dynamics of the ssRNAhelix junction, including the orientation and dynamics of the ssRNA tail relative to the reference helix, RDCs measured in the helix and ssRNA were subjected to an order tensor analysis (Losonczi et al 1999;Bailor et al 2007). Here, measured RDCs and an assumed local geometry for a given fragment are used to determine five elements of an order tensor describing the alignment of the fragment relative to the applied magnetic field (Saupe 1968); three Euler angles specify a principal ordering frame that describes the average orientation of the fragment relative to the applied magnetic field; a generalized degree of order (ϑ) (Tolman et al 2001) describes the degree of fragment alignment; and an asymmetry parameter (η = S yy − S xx /S zz ) describes the asymmetry of alignment.…”

Section: Global Structure and Sub-millisecond Motions From Rdcsmentioning

confidence: 99%

“…H splitting was ∼8 Hz in the presence of ∼8 mg/mL Pf1 phage (Asla Biotech). Idealized A-form structures were constructed using Insight II (Molecular Simulations) correcting the propeller twist angles from +15°to −15°using an in-house program, as previously described (Bailor et al 2007). The measured RDCs are listed in the Supplemental Data.…”

Section: Carbon Spin Relaxationmentioning

confidence: 99%

Structural dynamics of a single-stranded RNA–helix junction using NMR

Eichhorn

Al‐Hashimi

2014

RNA

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Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3 ′ end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, 13 C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A 6 -tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end. Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Our results reveal a surprising degree of structural and dynamic complexity at the ssRNA-helix junction, which involves a fine balance between order and disorder that may facilitate efficient pseudoknot formation on ligand recognition.

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“…Interesting details of RNA dynamics are also accessible from RDC measurements as shown in the work of Al-Hashimi [367,381]. Most RNAs which interact with proteins have more complex structures involving several helical stems and internal loops.…”

Section: Future Directionsmentioning

confidence: 93%