Fig. 1: Real-time observation of mammalian transcription elongation. a, Composite structural model of the human transcription EC with a set of elongation factors, as supplied in our system, decorating the Pol II surface. The model incorporates published structures for the mammalian EC (PDB 6TED), TFIIS (PDB 8A40), ELOF1 (PDB 8B3F) and a fitted AlphaFold prediction of IWS1 based on the position of Spn1 in the yeast EC (PDB 7XN7). b, Schematic of the single-molecule experimental setup. The starting location of the EC is marked by the ATTO647N-labeled RNA, while Cy3-labeled DNA probes that hybridize to the nascent RNA indicate active elongation. c, An example of transcription elongation by a single mammalian EC observed in real time. Top: kymograph showing the EC position on the DNA template, indicated by RNA (magenta) and probe (green) signals, as a function of time. Bottom: elongation trajectory extracted from the kymograph. Raw data (gray dots) were fitted to discrete linear segments (black line). Change points are marked as filled circles. The slopes for each segment are indicated, differentiating active elongation (blue) from pausing or stalling events (red). d, Eight additional examples of fitted elongation trajectories by individual mammalian ECs aligned by their starting positions. NTP, nucleoside triphosphate.
Fig. 7: Summary of the distinct roles that each elongation factor serves in mammalian Pol II transcription on DNA. The relative EC activity for each factor omission condition is color coded as indicated on the right. Clockwise from the top left: ΔTFIIS increases pausing but has a minimal effect on the elongation speed; ΔP-TEFb abrogates the phosphorylation of Pol II CTD and DSIF, which in turn destabilizes the binding of PAF1C, SPT6 and RTF1, thereby severely reducing the elongation speed; ΔPAF1C exerts the most direct negative impact on EC speed and also completely abolishes RTF1 recruitment; ΔRTF1 shifts the EC from high speed to medium speed; ΔSPT6 reduces EC speed by destabilizing PAF1C binding even though SPT6 itself does not directly activate EC; ΔDSIF exerts a dual effect on EC kinetics by reducing its pausing and also lowering its speed partially via the destabilization of RTF1 binding.
Transcribing Pol2
With elongation factors
Kinetic control
#ChromatinHaiku #SingleMolecule
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