Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II - PubMed (original) (raw)

Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II

Courtney Hodges et al. Science. 2009.

Abstract

RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for the regulation of eukaryotic transcription.

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Figures

Figure 1

Figure 1. Transcription through a nucleosome

(a) Geometry for the dual-trap optical tweezers experiments. (b) Representative trajectories of individual transcribing polymerases with or without the nucleosome at different ionic strengths. (c), (d), and (e) Probability of arrest or termination as a function of polymerase position on the DNA template at 300, 150, and 40 mM KCl respectively. Arrest is defined as a pause that lasts longer than 20 minutes or until the tether breaks. Data for transcription of bare DNA is in solid black, and nucleosome data is in semi-translucent colors. The shaded region represents the NPS.

Figure 2

Figure 2. Effect of the nucleosome on transcription dynamics

In each subplot, only traces that passed the NPS are considered. (a) Pause density with a nucleosome (dashed red line) and on bare DNA (solid black line). The pink shaded area represents the predicted pause density confidence interval at the nucleosome based on the model presented in the text. The gray shaded region is the confidence interval for pause density on bare DNA used in the model. Error bars are SEM. (b) Cumulative distributions of pause durations with (solid red line) and without (solid black line) a nucleosome present. Theoretical cumulative distributions for nucleosomal (pink dashed line) and non-nucleosomal (gray dashed line) pauses. (c) Pause-free velocities with (pink) and without (gray) a nucleosome with fits to normal distributions (solid lines). The predicted values based on the diffusive model with and without a nucleosome are shown as red and black circles, respectively.

Figure 3

Figure 3. Kinetic model of transcription through a nucleosome

The green area corresponds to on-pathway elongation (ke). The pink and blue areas represent off-pathway paused states where Pol II is backtracked; negative numbers indicate how many bases Pol II has backtracked from the elongation competent state, denoted by 0. The label u refers to the nucleosome being locally unwrapped (blue area), while w denotes the states where the nucleosome is wrapped in front of Pol II (pink area).

Figure 4

Figure 4. Histone transfer during transcription

(a) Force-extension curves of transcribed DNA. Pulling curves are shown in black and relaxation curves in red. (b) Frequency of histone transfer as a function of applied force during transcription.

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