Metal Ion Sensing\nby Tetraloop-like RNA Fragment:\nRole of Compact Intermediates with Non-Native Metal Ion–RNA\nInner-Shell Contacts

Divalent metal ions influence the folding and function\nof ribonucleic\nacid (RNA) in the cells. The mechanism of how RNA structural elements\nin riboswitches sense specific metal ions is unclear. RNA interacts\nwith ions through two distinct binding modes: direct interaction between\nthe ion and RNA (inner-shell (IS) coordination) and indirect interaction\nbetween the ion and RNA mediated through water molecules (outer-shell\n(OS) coordination). To understand how RNA senses metal ions such as\nMg<sup>2+</sup> and Ca<sup>2+</sup>, we studied the folding of a small\nRNA segment from the Mg<sup>2+</sup> sensing M-Box riboswitch using\ncomputer simulations. This RNA segment has the characteristics of\na GNRA tetraloop motif and interestingly requires the binding of a\nsingle Mg<sup>2+</sup> ion. The folding free energy surface of this\nsimple tetraloop system is multidimensional, with a population of\nmultiple intermediates where the tetraloop and cation interact through\nIS and OS coordination. The partially folded compact tetraloop intermediates\nform multiple non-native IS contacts with the metal ion. Thermal fluctuations\nshould break these strong non-native IS contacts so that the tetraloop\ncan fold to the native state, resulting in higher folding free energy\nbarriers. Ca<sup>2+</sup> undergoes rapid OS to IS transitions and\nvice versa due to its lower charge density than Mg<sup>2+</sup>. However,\nthe ability of Ca<sup>2+</sup> to stabilize the native tetraloop state\nis weaker, as it could not hold the loop-closing nucleotides together\ndue to its weaker interactions with the nucleotides. These insights\nare critical to understanding the specific ion sensing mechanisms\nin riboswitches, and the predictions are amenable for verification\nby nuclear magnetic resonance (NMR) experiments.