Abstract: RNA conformation is both highly dependent on and sensitive to the presence of charged ligands. Mono- and divalent ions stabilize the native fold of RNA, whereas other polyvalent cationic ligands can act to both stabilize or disrupt native RNA structure. In this work, we analyze the effects of two ligands, Mg (2+) and tobramycin, on the folding of S. cerevisiae tRNA (Asp) transcripts using single nucleotide resolution SHAPE chemistry. Surprisingly, reducing the Mg (2+) concentration favors a structural rearrangement in which the D- and variable loops pair. The tobramycin polycation binds to loops in tRNA (Asp) and induces RNA unfolding in two distinct transitions: the loss of tertiary interactions between the T- and D-loops followed by complete unfolding of the D-stem. Although Mg (2+) and tobramycin are relatively simple ligands, both modulate tRNA (Asp) folding in unanticipatedly complex ways, neither of which is consistent with simple hierarchical folding or unfolding of this RNA. Monitoring the structural consequences of ligand binding to RNA at single nucleotide resolution makes it possible to define intermediate structures that contribute to the complex energy landscapes often observed for RNA folding processes and lays the groundwork for a significantly improved understanding of the interactions between RNA and its solution environment.