Figure 2. Ligand binding by a natural singlet PRPP riboswitch.

(A) Sequence and secondary structure of the 106 nucleotide RNA derived from the purC gene of F. ignava. Data collected in B were used to determine regions of constant or reduced scission upon the addition of PRPP to in-line probing assays. Lowercase g indicates a guanosine nucleotide added to enable efficient transcription by T7 RNA polymerase. Arrowheads demark the approximate range of nucleotides with in-line probing data available as depicted in B. (B) Polyacrylamide gel electrophoresis (PAGE) analysis of the spontaneous RNA cleavage products generated during in-line probing of 5′-³²P-labeled 106 purC RNA. NR, T1 and ^–OH represent RNA undergoing no reaction, partial digest with RNase T1 (cleaves after guanosine nucleotides), or partial digest under alkaline conditions (cleaves after every nucleotide), respectively. Bands corresponding to selected G residues are annotated. In-line probing experiments contained either no ligand (–) or 1 mM of the compound indicated except guanine which was tested at 10 µM. Annotations 1 through 5identify prominent regions of the RNA that undergo structural stabilization in a PRPP-dependent manner. Data shown are representative of multiple experiments.