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

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1–4, 7) across a range of Mg(2+) concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg(2+), helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg(2+), the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg(2+)-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg(2+), the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg(2+). NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg(2+). We propose that Mg(2+) 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 interhelical 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.