Fig. 1: Impact of DNA Supercoiling and PQS Position on Transcription Rates Measured by Beacon Assay. | Nature Communications

Fig. 1: Impact of DNA Supercoiling and PQS Position on Transcription Rates Measured by Beacon Assay.

From: DNA supercoiling-mediated G4/R-loop formation tunes transcription by controlling the access of RNA polymerase

Fig. 1

a The schematic of beacon assay. Linearized and enclosed plasmids with identical sequence contexts are used to test the impact of DNA supercoiling on transcription. The DNA templates contain the T7 promoter and PQS, colored yellow and red, respectively. RNAP, colored gray, reads the DNA template and produces RNA, which anneals with the beacon, causing Cy3 signal enhancement. Real-time Cy3 signal measurements are obtained using a plate reader. b, c PQS orientation effect on RNA production. PQS locates 30 bp downstream of the Transcription start site (TSS) either in non-template (NT-PQS) or in template (T-PQS). As a control, random sequences are inserted (Control). b Linearized plasmid with NT- PQS (left, blue line) shows an enhanced RNA production rate, while T-PQS (left, black line) shows a decreased RNA production rate compared to Control (left, gray line). The plasmid with NT-PQS (right, blue line) does not exhibit enhanced RNA production compared to Control (right, gray line) and T-PQS (right, black line). c Initial RNA production rate is calculated from the early linear part of the curve in (b). Cy3 signals are normalized by that of each Control. Data are presented as mean ± SEM of 6 ~ 8 replicates of experiments. The exact data and P-values are provided in the Source data file. *P  <  0.05, **P  <  0.005 (two-sided unpaired t test) (d) PQS position effect on transcription. PQS positioned at 10, 30 and 60 bp downstream of TSS in non-template (NT-sp10, NT-sp30, NT-sp60). All linearized DNAs with NT-sp10, NT-sp30, and NT-sp60 are dashed faint, light, and dark purple lines, respectively. All plasmids with NT-sp10, NT-sp30, and NT-sp60 are solid faint, light, and dark purple lines, respectively. e Normalized Cy3 signals for -sp10, -sp15, -sp20, -sp25, -sp30, -sp35, -sp45 and -sp60 of NT-plasmid. The signals are normalized by the signal of Control. Data are presented as mean ± SEM of 5 replicates of experiments. The exact mean ± SEM and P-values are provided in the Source data file.

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