Modulation of Conformational Equilibria in the S-Adenosylmethionine (SAM) II Riboswitch by SAM, Mg2+, and Trimethylamine N-Oxide

The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg2+ ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites. Consequently, at low Mg2+ concentrations, Mg2+ binds preferentially to the open state, favoring its formation, but at high concentrations, Mg2+ binds preferentially to the compact state. The affinity of the riboswitch for SAM increases drastically with an increased level of binding of Mg2+ to the compact pseudoknot conformation. The effect of increasing concentrations of trimethylamine N-oxide (TMAO), a well-studied molecular crowding agent, on the conformation of the riboswitch and its affinity for SAM were also monitored by CD spectroscopy and measurement of diffusion. In the absence of added Mg2+, high concentrations of TMAO were found to induce a conformational change compatible with the formation of the pseudoknot form but have only a small effect on the affinity of the RNA for SAM.