Fig. 2: Characterization of NPOM as a transcriptional roadblock.
a Schematic representation of the DNA template used for the characterization of NPOM as a transcriptional roadblock. Only the 3′ extremity of the template is shown. NPOM-dT has been incorporated on the template strand at the 90th position. Upon UVA exposure, NPOM is cleaved, thus yielding an unmodified dT. b In vitro transcription experiments performed in the absence (−) or presence (+) or NPOM. Transcription was allowed to proceed for indicated amounts of time. The full-length (FL) and roadblocked (RB) species are shown. c Mapping of NPOM-roadblocked transcripts using 3′-OMe RNA sequencing reactions. The 3′-OMe nucleotide is shown above each lane and the nucleotide sequence is shown on the left. d Transcription assays performed in presence of streptavidin and NPOM roadblocks that were treated without (−) and with (+) washing steps. ECs were bound to Ni-NTA agarose beads for washing steps. Run-off transcription was used as a negative control. e NPOM-roadblocked and biotin/streptavidin-roadblocked ECs were subjected to GreB before and after UV irradiation. As a positive control, internal biotin/streptavidin roadblocked templates were used31. Roadblock (RB) and cleaved roadblock transcripts (cleaved RB) are shown on the left. The experiments were performed at least two times and variations were less than 5%.
