Thermodynamic examination of magnesium ion binding in the M‐box core 2 4×4 internal loop

Abstract

RNA binds to magnesium ions through diffuse and site‐specific interactions. Diffuse binding of magnesium ions to the backbone occurs in all RNA and is critical for the stabilization of RNA structure. Magnesium ions also bind to specific sites in particular RNA structures, often with direct Mg2+‐RNA bond formation. In this study, Isothermal Titration Calorimetry (ITC) was used to experimentally measure the enthalpy, binding constants, and Gibbs free energy of diffuse and site‐specific binding of magnesium ions to the core 2 of the Salmonella Typhimurium M‐box riboswitch. This Mg2+ binding bacterial riboswitch upregulates magnesium transporting proteins when intracellular ion concentrations are above an acceptable level. Crystal structure of the M‐box riboswitch RNA shows eight magnesium ion binding sites clustered in three cores. The core 2 of the M‐box riboswitch is made up of a 4×4 adenine‐rich internal loop that binds magnesium ions at residues A63 and A80. RNA constructs were designed to mimic the wild type core 2 and were also modified at positions A63 and A80. A helix construct, with the internal loop deleted, was also utilized. A statistical model for RNA‐Mg2+ binding was used to fit the ITC data to determine the thermodynamic parameters of diffuse and site‐specific magnesium ion binding to the RNA. The models used to fit ITC for ion binding will be presented. In general, we find that the diffuse binding is spontaneous, endothermic and entropically driven with binding constants in the low millimolar range. Site‐specific binding showed micromolar binding constants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.