Fig. 3: NDF−FACT condensate forms on a single Pol II–nucleosome transcription elongation complex.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Experimental set-up for single-molecule transcription experiments. This assay involves forming a stalled biotinylated-Pol II (bio-Pol II) elongation complex, which is ligated downstream to a molecular ruler and a single human nucleosome and tethered between two beads optically trapped through two DNA handles: one attached to the polymerase and the other to the upstream DNA. The addition of ribonucleotide triphosphates induced the polymerase to resume transcription and to move forward on the template, causing the upstream DNA to get extended. The instrument is operated in constant force mode, which causes the beads to move apart by 1 bp for each bp that the enzyme advances on the template. This ensures that the tension remains constant, allowing the movement of the beads to reflect the progress of the polymerase. b, Representative trajectories of individual Pol II transcribing through the molecular ruler (dotted lines correspond to the eight pauses) and human NPS, either alone or in the presence of NDF and FACT. Zoomed-in views of Pol II molecules transcribing the nucleosomes are shown on the right. c, The median residence time of Pol II at each bp across the nucleosome. The mean residence time is generated by averaging numerous single-molecule trajectories and serves as a quantitative description of the topography of the nucleosomal barrier. SHLs are shown. N = 20 biological replicates. d, A schematic of the C-trap experiments for simultaneous measurements of Pol II transcription in the optical tweezers channel and NDF−GFP and FACT−mCherry binding using a confocal microscope. Two Cy5 molecules in the SA−Biotin handle serve to mark the position of Pol II. e,f, Representative single-molecule traces of Pol II transcription (top) and fluorescence kymographs of NDF−GFP, FACT−mCherry and Cy5 signals of Pol II transcribing the molecular ruler and nucleosome template for the transcription experiment performed using a short template with 2 symmetrical 3.5 kb DNA handles (e) and transcription using a long template with asymmetric DNA handles (5.5 kb handle ligated to the upstream DNA and 3.5 kb handle tethered to Pol II) (f). The kymographs were obtained by continuously scanning the fluorescence lasers over the transcribing Pol II molecule until it reached the end of the template. The positions of the NPS entry, dyad and exit are indicated by dashed black lines. Scale bars, 1 µm.
