Force Spectroscopy of Add Adenine Riboswitch Aptamer Folding Reveals Multiple Intermediate and Misfolded States

Abstract

Riboswitches regulate genes expression through ligand-induced conformational changes. The folding of the add adenine riboswitch was investigated by repeatedly unfolding and refolding single aptamer molecules held under tension with optical tweezers. Multiple partially-folded intermediate states were identified from force-extension curves and folding trajectories at constant force, and characterized by measuring the associated molecular contour length changes, kinetics, and energetics. These states were correlated to essential structural components and interactions in the aptamer in a hierarchical folding pathway. In particular, the transition state for unfolding the ligand-bound aptamer involved the disruption of triplex helix-junction near the adenine binding pocket. The action of this riboswitch as a translational regulator (1) is found to be determined by the thermodynamic properties of the riboswitch, not by its kinetic properties as for the similar pbuE adenine riboswitch (2). In addition to on-pathway intermediates, several off-pathway, “misfolded” states were also observed and characterized. These results extend our understanding of the mechanics of RNA structure formation, the effects of multiple folding pathways, and the relation between folding and function in riboswitches. (1) Serganov, A., Yuan, Y.R., Pikovskaya, O., Polonskaia, A., Malinina, L., Phan, A.T., Hobartner, C., Micura, R., Breaker, R.R., and Patel, D.J. Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine-Sensing mRNAs. Chem. Biol. 11: 1729-1741 (2004). (2) Greenleaf, W.J., Frieda, K.L., Foster, D.A.N., Woodside, M.T. & Block, S.M. Direct Observation of Hierarchical Folding in Single Riboswitch Aptamers. Science 319, 630-633 (2008).