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. 2011 Nov 13;18(12):1394-9.
doi: 10.1038/nsmb.2164.

The Elongation Rate of RNA Polymerase Determines the Fate of Transcribed Nucleosomes

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Free PMC article

The Elongation Rate of RNA Polymerase Determines the Fate of Transcribed Nucleosomes

Lacramioara Bintu et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of yeast RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process through the distribution of transcribed nucleosome positions. Notably, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer and histone-histone dissociation quantitatively explains our observations and unifies them with results obtained from other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression.

Figures

Figure 1
Figure 1
Snapshots of transcription. AFM images of (a) stalled (no NTPs added) and (b) chased complexes (all four NTPs added). Only complexes that contain both the polymerase and nucleosome are included for analysis (unless otherwise specified); white arrows indicate Pol II in these complexes. (c) The height profile of an example complex (inset) is plotted along the DNA path as black circles. The Pol II and nucleosome heights are fit to Gaussians shown in purple and blue respectively. The free DNA segment lengths (black part of the fit) are defined as the lengths of the paths that start two standard deviations away from the centers of the proteins. (d) Schematic of the algorithm used to identify Pol II-nucleosome complexes. When Pol II (blue) is on the long arm of the nucleosome (gray) and its center has not yet reached the middle of the DNA template (green vertical line), we deem the complex untranscribed. When Pol II has passed the middle line of the template, and the Pol II and nucleosome edges are within 5 nm or less of each other, the complexes are tagged as intermediate. When Pol II is on the short arm of the nucleosome, the complex is tagged as transcribed.
Figure 2
Figure 2
Nucleosome position. (a) The length of the downstream free DNA segments for untranscribed nucleosomes (red) and complexes without Pol II (blue). (b) The length of the upstream free DNA segment for transcribed nucleosomes (red) and complexes without Pol II (blue).
Figure 3
Figure 3
DNA looping during histone transfer. (a) AFM images of complexes where the histones contact both the upstream and downstream DNA. (b) Free DNA length in the presence of NTPs (all concentrations) in complexes where Pol II is in the process of transcribing the nucleosome (the length of DNA between Pol II and the nucleosome is less than 5 nm apart) and (c) complexes where Pol II has started transcription, but has not yet reached the nucleosome. Insets show the presumed structures of each population, with Pol II in blue, the DNA in black, and the histones in brown; the pink shading reflects the apparent broadening of the molecules due to the geometry and size of the AFM tip. Numbers indicate the mean and standard deviations of the total free DNA lengths.
Figure 4
Figure 4
Transcription leads to hexamer formation. (a) Images illustrating change in nucleosome reduction upon transcription. The nucleosomes with reduced height are shown next to normal-sized nucleosomes for comparison. Histograms of nucleosome heights for (b) untranscribed nucleosomes and (c) transcribed nucleosomes at all NTPs concentrations show the appearance of subnucleosomal particle with reduced height (fit by the red curve). (d) Heights of octamers (blue fit) compared with tetramers (green fit). (e) Height of nucleosomes destabilized by incubation in high salt (1 M KCl). We identify the three peaks as tetramers (green), hexamers (red) and octamers (blue).
Figure 5
Figure 5
Histone transfer outcome depends on the speed of transcription. Heights of transcribed nucleosomes at (a) 100 µM NTPs, (b) 200 µM NTPs, and (c) 1000 µM NTPs. Nucleosome heights for molecules where Pol II has not passed the nucleosome in (d) 100 µM NTPs, (b) 200 µM NTPs, and (c) 1000 µM NTPs. The continuous curves represent Gaussian fits to the data. Insets are showing transcribed nucleosomes (top) and untranscribed nucleosomes (bottom).
Figure 6
Figure 6
Histone transfer model. (a) Kinetic scheme of transcription and histone transfer. Pol II in blue, H2A–H2B dimers in red, H3–H4 dimers in brown, DNA in black. (b) Hexamer (red) and octamer (blue) transfer probabilities, as well as bare DNA formation (green) are plotted as a function of the net nucleosome unwrapping rate during elongation (kue). Experimental data are shown as circles; the shaded areas represent the model predictions, with the width reflecting uncertainties in kt and N, as indicated in the text. The black line marks the elongation rate for the faster polymerase Pol III.

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