Chromatin Buffers Torsional Stress During Transcription

Abstract

Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA. In this work, we developed methods to visualize Pol II rotation of DNA during transcription and determine how torsion slows down the transcription rate. We found that Pol II stalls at ± 9 pN·nm torque, nearly sufficient to melt DNA. The stalling is due to extensive backtracking, and the presence of TFIIS increases the stall torque to + 13 pN·nm, making Pol II a powerful rotary motor. This increased torsional capacity greatly enhances Pol II's ability to transcribe through a nucleosome. Intriguingly, when Pol II encounters a nucleosome, nucleosome passage becomes more efficient on a chromatin substrate than on a single-nucleosome substrate, demonstrating that chromatin efficiently buffers torsional stress via its torsional mechanical properties. Furthermore, topoisomerase II relaxation of torsional stress significantly enhances transcription, allowing Pol II to elongate through multiple nucleosomes. Our results demonstrate that chromatin greatly reduces torsional stress on transcription, revealing a novel role of chromatin beyond the more conventional view of it being just a roadblock to transcription.

Figure 1.

Visualizing Pol II rotation and stalling under torsion. a. An experimental configuration to track Pol II rotation of DNA during transcription using an angular optical trap (AOT). Pol II is torsionally anchored on the surface of a coverslip, and its downstream DNA is torsionally attached to the bottom of a quartz cylinder, which is trapped by the AOT. Pol II elongation generates (+) supercoiling in the downstream DNA. The cylinder rotates to follow Pol II rotation to maintain a constant torque in the DNA. The right panel shows that the extraordinary axis of the cylinder tends to align with the linear polarization of the input trapping beam. The orientation of this axis is accurately detected and informs the angular orientation of the DNA attached to the cylinder. b. A representative real-time trajectory of Pol II rotation of DNA during transcription under a +3.2 pN·nm resistance torque. The scale bar shows the conversion to base pair position of Pol II (or nucleotides transcribed). Regions of Pol II steady rotation are marked red, and regions of pausing are marked black. See Supplementary Movie 1 for the corresponding video. c. Pol II stall torque measurements using the AOT. The two cartoons illustrate the experimental configurations to stall Pol II against (−) torsion upstream (left) and (+) torsion downstream (right), mimicking the “twin-supercoiled-domain” model of transcription. The measured stall torque distributions are shown beneath the corresponding cartoon, with the mean and SEM indicated. d. Representative traces of Pol II backtracking upon stalling under (+) torsion. Different colors represent different traces. Pol II forward translocates and stalls under increased (+) torque. Upon stalling, Pol II backtracks, as evidenced by the reverse motion.

Figure 2.

TFIIS up-regulates Pol II’s ability to transcribe under torsion. a. Experimental configuration to monitor transcription activity under torsion via magnetic tweezers. Pol II is torsionally anchored to the surface of a magnetic bead, while its downstream DNA is torsionally anchored to the surface of a coverslip. The magnetic bead can be used to manipulate the torsional state of the DNA and the force on the DNA. b. Example traces of Pol II elongation during a torque-jump experiment. Pol II transcription starts at a torque of low value before jumping to a higher value. Since the DNA is buckled, the force on the DNA informs the torque in the DNA (Supplementary Fig. 4d). The two plots show the Pol II position from the transcription start site (TSS) without and with TFIIS. c. The mean trajectory of Pol II elongation under different torques. All traces were aligned at the start of the torque jump (t = 0), with the shaded region for each curve representing 30% of the standard deviation. Without TFIIS: N = 87, 72, and 42 for 0, 6, and 10 pN·nm, respectively. With TFIIS: N = 36, 72, and 61 for 0, 6, and 10 pN·nm, respectively. d. Torque-velocity relation. Pause-free velocities are shown without and with TFIIS. The error bars represent the SEM, with each data point collected from N = 27–87 traces. e. Active fraction after the torque jump. The active fraction is the fraction of traces remaining active at a given torque. Each curve is fit with a decaying function (Methods) to obtain the critical torque τ_c, at which 50% of traces are active. The fit values and uncertainties without and with TFIIS are also shown.

Figure 3.

A real-time assay of tracking Pol II transcribing through nucleosomes under torsion via magnetic tweezers. a. Experimental configurations. Pol II is torsionally anchored to the surface of a magnetic bead. The DNA downstream of Pol II could contain a single nucleosome (left) or a nucleosome array (right) (Supplementary Fig. 3b) and was torsionally anchored to the surface of a coverslip. Despite having different nucleosomes, the two templates are identical in their overall length and in the sequence up to the first nucleosome encountered. b. Representative trajectories of Pol II transcribing through nucleosomes under torsion. The left panels show traces from the template containing a single nucleosome. The right panels show traces from the template containing a nucleosome array. The measured extension is converted to the Pol II position from the TSS for each trace (Methods). Each shaded region represents an expected nucleosome position. c. Pol II dwell time distribution at a nucleosome encounter. The dwell time at each position after Pol II encountering a nucleosome is calculated from the Pol II trajectories using a “first-passage” method (Methods). The dark grey shaded region indicates the location of the nucleosome positioning element for the first nucleosome encountered.

Figure 4.

Chromatin buffers torsional stress to facilitate transcription. a. Mean trajectories of Pol II transcription through a nucleosome. All traces were aligned when Pol II reached the entry of the 1^st nucleosome encountered (t = 0), with the shaded regions representing 30% of the standard deviation. The left panel shows data from the single-nucleosome template (left; N = 86 for -TFIIS, N = 215 for +TFIIS). The right panel shows data from the first nucleosome on the chromatin template (right; N = 99 for -TFIIS; N = 302 for +TFIIS). b. Pol II nucleosome-passage rate. The mean passage rates under torsion were obtained from data shown in a, with the torque value being the torque experienced by Pol II when encountering the nucleosome dyad (Methods). The mean passage rates under zero-torsion of the single-nucleosome template were obtained from data shown in Supplementary Fig. 8. The error bars represent the SEMs.

Figure 5.

Torsion modulates nucleosome passage. a. Experimental configuration of transcription through chromatin in the presence of topo II. This configuration is identical to that used in Fig. 3a (right), except for having topo II present in the assay. All experiments were carried out in the presence of TFIIS. b. Explanation of the method used to determine the number of nucleosomes passed. The extension-turns relation is determined by the number of nucleosomes and the DNA length between Pol II and the bead. Both the width and height of the extension-turns curve decrease after transcription. Shown is an example trace where we measured 49 nucleosomes before transcription and 47 nucleosomes after transcription, indicating Pol II having passed 2 nucleosomes during transcription. c. The number of nucleosomes Pol II passes through during transcription versus transcription time. The error bars represent the SEM, with each data point collected from N = 14–33 traces. d. Experimental configuration to modulate torsion using the magnetic bead during transcription through chromatin. This configuration is similar to that used in Fig. 3a (right), except that the magnet bead is rotated at a constant rate. All experiments were carried out in the presence of TFIIS. e. Mean trajectories of Pol II transcription through a nucleosome. All traces were aligned when Pol II reached the entry of the 1^st nucleosome encountered (t = 0), with the shaded regions representing 30% of the standard deviation. Shown are examples of magnet rotation rates at +0.05 turns/s (hindering transcription) (N = 30) and −0.13 turns/s (assisting transcription) (N = 49). f. Pol II nucleosome-passage rates at different magnet rotation rates. The mean rates for the passage of the 1^st nucleosome encountered are shown, with the error bars representing the ± SEMs, with each data point collected from N = 25–65 traces.