Structural analysis of the Adenine Riboswitch, which is a bacterial regulatory RNA that binds Adenine and controls gene expression [35, 37]. The RNA adopts two major conformations, the “On” state (Adenine bound) forms three stem loops (P1, P2 and P3), while in the “off state” the site of translation initiation (3’ end of the UTR, near the start codon) is structured effectively disrupting translation initiation. A.) Boltzmann suboptimal sampling of the ensemble of possible RNA conformations (as predicted by sFold) projected onto the first two principal components of structure space as determined by a Manhattan distance metric evaluation of the ensemble. Each dot in the diagram is one alternative structure. Representative structures adorn the diagram, and the aptamer domain of the Riboswitch is highlighted in light magenta. The Riboswitch is predicted to adopt four structures, characterized by green, purple, cyan and red dots. The “on” and “off” states of the Riboswitch to the green and magenta cluters, respectively. B.) Boltzmann sampling of the structural ensemble for the C77G containing sequence which indicates a significant shift in partitioning towards the cyan and red conformations. C.) Boltzmann sampling for the U39A mutation which is predicted to have no effect on the partitioning compared to WT. D.) Experimental validation using SHAPE chemistry of the predictions made in A-C, showing that the C77G mutation disrupts the structure of the RNA in a manner consistent with an increase in the population of the cyan cluster.