Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

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2018-07-16

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Abstract

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of non-coding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1, 2, 3, 4 and 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+ (25 mM monovalent salt), helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population < 15%) whereas 1-bulge and 2-bulge motifs adopt predominantly stacked conformations (stacked population > 74%). The 2-bulge motif is biased toward linear conformations and increasing the bulge length leads to broader inter-helical distributions and structures that are on average more kinked. In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population > 84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition does not vary substantially (within 3-fold) with bulge length. In the absence of Mg2+, the difference between the free energy of inter-helical coaxial stacking across the bulge variants is estimated to be ≈2.9 kcal/mol, based on an NMR chemical shift mapping approach, with stacking being more energetically disfavored for the longer bulges. This difference decreases to ≈0.4 kcal/mol in the presence of 3 mM Mg2+ NMR residual dipolar coupling and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+ We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated inter-helical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

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10.1261/rna.066258.118

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Merriman, Dawn K, Jiayi Yuan, Honglue Shi, Ananya Majumdar, Daniel Herschlag and Hashim M Al-Hashimi (2018). Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics. RNA (New York, N.Y.). pp. rna.066258.118–rna.066258.118. 10.1261/rna.066258.118 Retrieved from https://hdl.handle.net/10161/17418.

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Al-Hashimi

Hashim Al-Hashimi

Adjunct Professor in the Department of Biochemistry

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