Fig. 3. TPP sensing is preferentially achieved cotranscriptionally by the tbpA riboswitch.

a RNase H probing experiments monitoring TPP binding when performed cotranscriptionally (Co-trx) or post-transcriptionally (Post-trx). Control reactions (Ctrl) done in the presence of RNase H (RH) show a cleaved product (P) and the uncleaved full-length (FL) transcript without TPP. Cotranscriptional binding was determined by adding TPP before transcription initiation and performing RNase H cleavage during transcription. Post-transcriptional measurements were achieved by performing transcription without TPP, which was followed by the addition of rifampicin, TPP and RNase H cleavage assays. b Quantifications of cotranscriptional and post-transcriptional binding assays. Experiments were fitted to a single-exponential assays and the values of the calculated rates are indicated. c K_switch measurements for the wild-type tbpA riboswitch done in the presence of increasing TPP concentrations. The full-length (FL) and the cleave product (P) are shown on the left of the gel. d Quantification of the K_switch experiments done when using 50 µM or 500 µM NTP. The K_switch value is increasing upon decreasing the NTP concentrations. The average and the standard deviations are shown for each data point.