Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding

Abstract

Riboswitches are gene-regulating RNAs that are usually found in the 5′-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested monoand divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg2+, Ca2+, and Mn2+ facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how iondependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected. Published by Cold Spring Harbor Laboratory Press. Copyright © 2010 RNA Society.