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. 2013 Jun 28;340(6140):1580-3.
doi: 10.1126/science.1235441.

Transcription under torsion

Jie Ma  1 , Lu Bai, Michelle D Wang
Affiliations

Transcription under torsion

Jie Ma et al. Science. .

Abstract

In cells, RNA polymerase (RNAP) must transcribe supercoiled DNA, whose torsional state is constantly changing, but how RNAP deals with DNA supercoiling remains elusive. We report direct measurements of individual Escherichia coli RNAPs as they transcribed supercoiled DNA. We found that a resisting torque slowed RNAP and increased its pause frequency and duration. RNAP was able to generate 11 ± 4 piconewton-nanometers (mean ± standard deviation) of torque before stalling, an amount sufficient to melt DNA of arbitrary sequence and establish RNAP as a more potent torsional motor than previously known. A stalled RNAP was able to resume transcription upon torque relaxation, and transcribing RNAP was resilient to transient torque fluctuations. These results provide a quantitative framework for understanding how dynamic modification of DNA supercoiling regulates transcription.

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Figures

Figure 1
Figure 1. Stall torque experiments
(A) (Top) A cartoon depicting the “twin-supercoiled domain” model (1). (Bottom) Experimental configuration that mimics the “twin-supercoiled domain” model for transcription against (−) supercoiling upstream or (+) supercoiling downstream. (B) A representative set of data for downstream stall torque measurements. After the introduction of NTPs, the force on the DNA was clamped at a low value while DNA was mechanically unwound to form a (−) plectoneme. Subsequent translocation of RNAP neutralized the (−) plectoneme (➀ and ➁) and resulted in (+) plectoneme formation (➂). The force clamp was then turned off (➃). RNAP translocation increased the force (directly measured) and the corresponding torque (derived) (11) until reaching a stall (< 1 bp/s for 20–50 s). Data were filtered: extension to 200 Hz (black) and 1 Hz (red), and force to 40 Hz (black) and 1 Hz (red). The RNAP template position is defined as the distance of RNAP from the transcription start site (in bp).
Figure 2
Figure 2. Transcription stalling and resumption
(A) The distribution of the measured downstream stall torques. The smooth blue curve is a fit with a Gaussian function, yielding a mean of 11.0 ± 3.7 pN•nm (mean ± SD). (B) The distribution of measured upstream stall torques. The smooth curve is a fit with a Gaussian function assuming that the peaked fraction generated torques of at least 10 pN•nm, yielding a mean of 10.6 ± 4.1 pN•nm (mean ± SD). (C) Example traces showing RNAP reverse translocation upon stalling. Both axes are shifted for clarity. For each trace, the arrow indicates the entry into a stall. (D) Fraction of RNAPs that resumed transcription after torque release versus time. After stalling, torque on RNAP was relaxed and transcription was detected by an experiment similar to that shown in ➀ of Figure 1B.
Figure 3
Figure 3. Transcription response to a transient torque pulse
(A) (Top) A cartoon illustrating steps of the “torque pulse” experiments and (Bottom) representative traces of data. RNAP initially transcribed under a low downstream torque of approximately +7 pN•nm, and then was subjected to a higher torque pulse for either 5 s or 0.5 s before restoration of the initial low torque. Traces 1 and 4 are controls. The extension and time axes are shifted for clarity. (B) The probability of maintaining active transcription during the 5 s torque pulse. The blue solid line is a fit to a Boltzmann function): f = 1/[1+e(τ−τc)/τ0], where τc is the characteristic cutoff torque. (C) The probability of resuming transcription immediately (within 5 s) after the torque pulse.
Figure 4
Figure 4. Determination of transcription torque-velocity relationship
(A) A representative set of data for transcription measurement under a constant torque. Transcribing RNAP, under a small and constant tension of 0.15 pN, was subjected to multiple cycles of resisting and assisting torque. For each cycle, the downstream DNA was mechanically unwound to remove any (+) plectoneme (➀) and create a (−) plectoneme (➁). Subsequent RNAP transcription was assisted by the (−) DNA supercoiling (➂), until the generation of (+) supercoiling, which hindered transcription (➃). In the presence of a plectoneme, the torque on the DNA was constant for a given force (9) (fig. S3) and RNAP velocity was derived from the slope of the extension versus time curve (11). Also, we define a resisting torque to be (+) and an assisting torque to be (−). Data were filtered to 200 Hz (blue and red) and 1 Hz (grey). (B) Representative transcription traces under a torque of +7.5 pN•nm. Continuous transcription (green smoothed data) was interrupted by pauses (red smoothed data), each of which is indicated by a red line. (C) Transcription torque-velocity relationship. Transcription velocity was obtained by weighting each transcript position equally and the resulting velocity reflected primarily transcription rates between pauses (11, 29). (D) Pause density (top) and duration (bottom) as a function of torque. A pause is defined as having a duration of ≥0.2 s at a given nucleotide position (11). Zero-torque data (fig. S8) had lower sensitivity to transcription due to lack of plectoneme in DNA, precluding detection of pauses of 0.2–2 s in duration, and were thus excluded from pause analysis.
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