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

ABSTRACT: 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 A6-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.

SUBMITTER: Eichhorn CD

PROVIDER: S-EPMC4024633 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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

Eichhorn Catherine D CD Al-Hashimi Hashim M HM

RNA (New York, N.Y.) 20140417 6

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 ord ...[more]

PMID: 24742933

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Project description:A growing number of studies employ time-averaged experimental data to determine dynamic ensembles of biomolecules. While it is well-known that different ensembles can satisfy experimental data to within error, the extent and nature of these degeneracies, and their impact on the accuracy of the ensemble determination remains poorly understood. Here, we use simulations and a recently introduced metric for assessing ensemble similarity to explore degeneracies in determining ensembles using NMR residual dipolar couplings (RDCs) with specific application to A-form helices in RNA. Various target ensembles were constructed representing different domain-domain orientational distributions that are confined to a topologically restricted (<10%) conformational space. Five independent sets of ensemble averaged RDCs were then computed for each target ensemble and a "sample and select" scheme used to identify degenerate ensembles that satisfy RDCs to within experimental uncertainty. We find that ensembles with different ensemble sizes and that can differ significantly from the target ensemble (by as much as ∑Ω ∼ 0.4 where ∑Ω varies between 0 and 1 for maximum and minimum ensemble similarity, respectively) can satisfy the ensemble averaged RDCs. These deviations increase with the number of unique conformers and breadth of the target distribution, and result in significant uncertainty in determining conformational entropy (as large as 5 kcal/mol at T = 298 K). Nevertheless, the RDC-degenerate ensembles are biased toward populated regions of the target ensemble, and capture other essential features of the distribution, including the shape. Our results identify ensemble size as a major source of uncertainty in determining ensembles and suggest that NMR interactions such as RDCs and spin relaxation, on their own, do not carry the necessary information needed to determine conformational entropy at a useful level of precision. The framework introduced here provides a general approach for exploring degeneracies in ensemble determination for different types of experimental data.

Dataset Information

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

Publications

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

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