Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent

Abstract

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions, in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state although Mg 2+ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and non-specifically bound to the RNA, as predicted by the counterion condensation (CIC) theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained Three Site Interaction (TIS) model, with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg 2+ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic non-monotonic shape fluctuations in the ribozyme as it folds with increasing Mg 2+ or Ca 2+ concentration.

Charge density of cation determines inner versus outer shell coordination to phosphate in RNA

“…Accurate simulations using coarse-grained models of ribozymes in explicit monovalent and divalent cations 85 is computationally demanding. 25,26 In order to simplify the problem, while still retaining a high level of accuracy in predicting the RNA folding thermodynamics and kinetics, we developed a theory to treat the electrostatic interactions involving monovalent ions implicitly, while treating the many body effects due to divalent ions explicitly. 38 Briefly, we assumed that the screening effect due to monovalent cations, especially when present at high concentrations, can be described by the classical Debye-Huckel (DH) theory.…”

“…2,25 40 Recently, simulations of a large ribozyme and several other RNA constructs, in the presence of both monovalent and divalent ions, have shown that the ions have a strong propensity to bind to nucleotides in a site-specific manner, and not uniformly. [25][26][27] The irregular shape of the RNA plays an important role in determining where the ions bind, as the ions are naturally drawn to the highly negatively charged pockets.Other studies, focusing only on the folded states or well-defined complexes involving RNA, have shown 45 that the site-specifically bound Mg 2+ ions to various RNA molecules are stable. [28][29][30][31][32][33] These studies did not consider ion-driven folding of RNA from a completely unfolded state, which is the subject of our interest here and elsewhere.…”

“…[28][29][30][31][32][33] These studies did not consider ion-driven folding of RNA from a completely unfolded state, which is the subject of our interest here and elsewhere. 25,26 In order to develop a quantitative theory of RNA folding, effects of ions and the associated conformational changes they induce in RNA must be treated on equal footing.Conceptually, specific association of divalent ions to RNAs could be categorized into two binding types: 50…”

“…2,25 40 Recently, simulations of a large ribozyme and several other RNA constructs, in the presence of both monovalent and divalent ions, have shown that the ions have a strong propensity to bind to nucleotides in a site-specific manner, and not uniformly. [25][26][27] The irregular shape of the RNA plays an important role in determining where the ions bind, as the ions are naturally drawn to the highly negatively charged pockets.…”

“…Divalent ion-driven folding of RNA is a strongly interacting many body problem because the binding of several Mg 2+ ions in a specific and correlated manner is needed to obtain a stable folded structure. 25,26 Already at the single ion level, the interplay between the inner and outer sphere coordination manifests itself. In a pioneering series of studies conducted nearly thirty years ago, Rossky and coworkers used 60 extended reference interaction site model (RISM) theory and simulations to show that the potential of mean force (PMF) between Na + and phosphate exhibits the two binding modes mentioned above.…”