The effect of ligand binding on RNA susceptibility to spontaneous breakdown. In this experiment, a transcript containing just the TPP riboswitch aptamer sequence was incubated under slightly alkaline conditions in the presence of increasing concentrations of TPP. The leftmost lane (NR) of the polyacrylamide gel contains untreated RNA; the dark band indicates the size of the full-length RNA. The next two lanes show the fragments generated by partial digestion of the RNA with either riibonuclease T1 (T1) or strong alkali (-OH) in the absence of TPP. Many sites on the RNA are susceptible to cleavage when TPP is not present, indicating that the RNA does not form a stable three-dimensional structure. As the remaining lanes show, when the RNA is incubated for long periods of time under slightly alkaline conditions, fewer fragments are generated as the TPP concentration is increased (arrows). This demonstrates that TPP promotes formation of secondary and tertiary structures that protect certain residues from spontaneous cleavage.