Metal Ion Sensing by Tetraloop-like RNA Fragment: Role of Compact Intermediates with Non-Native Metal Ion–RNA Inner-Shell Contacts

Divalent metal ions influence the folding and function of ribonucleic acid (RNA) in the cells. The mechanism of how RNA structural elements in riboswitches sense specific metal ions is unclear. RNA interacts with ions through two distinct binding modes: direct interaction between the ion and RNA (inner-shell (IS) coordination) and indirect interaction between the ion and RNA mediated through water molecules (outer-shell (OS) coordination). To understand how RNA senses metal ions such as Mg²⁺ and Ca²⁺, we studied the folding of a small RNA segment from the Mg²⁺ sensing M-Box riboswitch using computer simulations. This RNA segment has the characteristics of a GNRA tetraloop motif and interestingly requires the binding of a single Mg²⁺ ion. The folding free energy surface of this simple tetraloop system is multidimensional, with a population of multiple intermediates where the tetraloop and cation interact through IS and OS coordination. The partially folded compact tetraloop intermediates form multiple non-native IS contacts with the metal ion. Thermal fluctuations should break these strong non-native IS contacts so that the tetraloop can fold to the native state, resulting in higher folding free energy barriers. Ca²⁺ undergoes rapid OS to IS transitions and vice versa due to its lower charge density than Mg²⁺. However, the ability of Ca²⁺ to stabilize the native tetraloop state is weaker, as it could not hold the loop-closing nucleotides together due to its weaker interactions with the nucleotides. These insights are critical to understanding the specific ion sensing mechanisms in riboswitches, and the predictions are amenable for verification by nuclear magnetic resonance (NMR) experiments.