Fig. 3: The impact of PQS in transcription regulation diminishes with reduced DNA supercoiling.
a Beacon assay (described in Fig. 1) conducted with DNA templates in various topological states. Plasmid-NT-sp10, NT-sp30, and control samples were pre-treated with topoisomerase I (Topo I) at different concentrations to create varying degrees of superhelicity. The gel image on the left shows NT-sp10 treated with 10 units (highest), 5 units (high), 3.3 units (mid), and 2.5 units (low) of Topo I, representing a gradient of supercoiling levels. For further details, see Supplementary Fig. 4 and the Methods section. In vitro transcription reactions are conducted with the pre-treating DNAs with Topo 1 (right). The more orange-colored the line, the less supercoiled the DNA construct is. (b) Bar graph of (a) displaying the normalized Cy3 signal increase for each DNA construct. Bars represent the mean ± SEM from three independent experiments. c In vitro transcription reactions are conducted with increasing concentrations of Topo 1, which gradually relaxes negative supercoiling during transcription. d Bar graph of (d) displaying the normalized Cy3 signal increase for each DNA construct. For (b) and (d), data are presented as mean ± SEM of 3 ~ 4 replicates of experiments. The exact data and P-values are provided in the Source data file. *P < 0.05, **P < 0.005, ***P < 0.0005, NS: nonsignificant (two-sided unpaired t- test).
