Figure 4. The pbuE riboswitch requires a transcriptional context to bind adenine.

(A) Single-round in vitro transcriptions of the pbuE riboswitch using the B. subtilis RNA polymerase (RNAP). Top, transcription reactions performed as a function of 2,6-diaminopurine (DAP). Readthrough (RT) and terminated (T) products are indicated on the right. Bottom, percentage of readthrough products plotted as a function of DAP concentration. The line shows a two-state model from which a T₅₀ value of 2.1±0.2 µM DAP was calculated. (B) Single-round in vitro transcriptions of the pbuE riboswitch using the E. coli RNAP. Top, transcription reactions performed as a function of DAP. Bottom, percentage of readthrough products plotted as a function of DAP concentration. A T₅₀ value of 0.5±0.1 µM was obtained from the fitting. The insert shows transcription reactions performed as a function of adenine concentration. A T₅₀ value of 2.3±0.3 µM was calculated for adenine. (C) Single-round in vitro transcriptions performed in presence of various ligands. Reactions were performed in absence (−) of ligand and in presence of 10 µM adenine (Ade), 2,6-diaminopurine (DAP), 2-aminopurine (2AP), hypoxanthine (Hyp) and guanine (Gua). Percentages of readthrough (RT) products are indicated below the gel. (D) Thermal denaturation of the pbuE aptamer in absence and in presence of 10 µM DAP. Melting temperatures (T_m) were evaluated by determining the temperature required to obtain half of the melting transition (normalized OD = 0.5) [79], [80]. (E) Single-round in vitro transcriptions performed as a function of the P1 stem elongation. The P1 stem was elongated by 2, 3, 5, 8 and 10 bp and resulting constructs were assayed using single-round in vitro transcriptions. The wild-type pbuE riboswitch contains a P1 stem of 5 bp. (F) Single-round in vitro transcriptions performed as a function of P1 stem destabilization. The P1 stem was shortened by 1, 2 and 3 bp and resulting constructs were assayed using single-round in vitro conditions.