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First published online 21 May 2008
doi: 10.1242/jcs.027250

Journal of Cell Science 121, 1999-2007 (2008)
Published by The Company of Biologists 2008
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RNA polymerase II activity is located on the surface of protein-rich transcription factories

Christopher H. Eskiw*, Alexander Rapp, David R. F. Carter and Peter R. Cook{ddagger}

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK


Figure 1
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  		Fig. 1. Experimental approach. (A) Cartoon illustrating the principles that underly ESI. (B) Conventional EM image. HeLa cells were permeabilised, polymerases allowed to extend transcripts in BrUTP, BrRNA indirectly immunolabelled with gold particles, and 70 nm sections stained with uranyl acetate. The nucleolus and heterochromatin are heavily stained with the heavy metal, to appear black-on-white (as many electrons are scattered and so not detected). Inset, three gold particles (marking nascent BrRNA) also scatter electrons to appear dark. (C) ESI (P maps) of unstained cells (i.e. lacking uranyl acetate). Left, cell prepared as in B, but without staining. Right, control that was fixed directly, and was not permeabilised, immunolabelled or stained. P-rich structures, such as heterochromatin, appear white-on-black, and permeabilisation has little effect on morphology. Scale bars: 1 µm (inset in B, 50 nm).

 

Figure 2
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  		Fig. 2. Imaging sites (marked by BrRNA) using ESI. HeLa cells were permeabilised, nascent transcripts extended in BrUTP, cells fixed and BrRNA indirectly immunolabelled with gold particles; after sectioning (70 nm), elemental maps were collected by ESI. Arrows indicate clusters of particles. (A-D) Four views of one section. The zero-loss image reveals little other than the cluster of particles marking BrRNA, but P and N maps are complex; in the merge, the grey scale for the zero-loss image is inverted, and particles (now white) mark a N-rich structure. (E-I) Similar merges of other structures labelled with particles; all structures are N-rich. Scale bars: 100 nm. (J) After run-on ± actinomycin D, the number of lone (brown bars) and clustered particles (blue bars) in 15 images (total area 44 µm2) as in D were counted; the inhibitor reduces the number of particles indicating that most mark nascent RNA. As a cluster typically contains 2.9 particles (123 sites analysed), the 2D density of sites marked by clusters is 1.2 sites/µm2, which is equivalent to a 3D density of 10.3 sites/µm3. Errors bars represent s.d.

 

Figure 3
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  		Fig. 3. Site perimeter, diameter and atomic content. (A-D) Perimeters of N-rich structures are mapped by subtracting the intensity in each pixel (1.35 nm2) in a P map (B) from its counterpart in the N map (A); this process is equivalent to removing chromatin from the image (as nucleosomes yield similar intensities in both channels; Table 1). Next, the resulting image is binarised, holes within (N-rich) structures filled, structures `eroded' by 1 pixel (1.35 nm2) twice (to remove small structures), and then `dilated' twice to restore structures to the original size. The resulting masks (C) outline various structures; those with diameters >40 nm were selected (justifiable because all clusters are associated with N-rich structures of >40 nm). The three remaining structures are coloured purple, blue, and orange, and their perimeters are overlaid over the zero-loss/P/N merge (D). One perimeter bounds an active transcription site marked by three particles, another (purple) could ring a site (it possesses the appropriate N:P ratio) but is not labelled with gold, whereas a third (blue) cannot be a site (it has the N:P ratio of a coiled body; not shown). (E) Frequency of site diameters. Sites were selected by choosing those marked by clusters, and then drawing perimeters around associated N-rich structures (n=69) as in D. Major and minor (orthoganol) axes of structures were measured, and diameters calculated (assuming underlying structures are spheres) and binned (20-nm bins of 0-19, 20-39, 40-59 nm, etc.). Diameters were corrected for effects of sectioning, which can remove polar caps (such correction gives rise to sites of <40 nm). Uncorrected and corrected diameters were normally distributed (Materials and Methods) about means of 96 and 87 nm, respectively. If many small sites were being missed, the distribution would be highly skewed to the left with many more 40-60 nm structures being seen. (F-I) N and P content were determined using merges such as that in D with nucleosomes (arrowheads) as references. Dotted lines: perimeters of N-rich structures marked by gold particles. Scale bars: 100 nm.

 

Figure 4
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  		Fig. 4. Identifying sites by correlative microscopy and in unpermeabilised cells. (A-E) Permeabilised HeLa cells were allowed to extend nascent transcripts in BrUTP, and the resulting BrRNA indirectly immunolabelled with Cy3; cells were then embedded, sectioned (70 nm), and images of the same sections collected by fluorescence and electron microscopy. (A) Merge of fluorescence and –155 eV EM images; sites containing nascent BrRNA (yellow) are scattered throughout the nucleoplasm (grey). Insets, selected areas shown in B-E. (B) Collage of ESI merges (P, red; N, green) of the same region. (C-E) Typical ESI merges (P, red; N, green) of regions shown in insets in A and B. (F-H) Cells were directly fixed, sectioned (without immunolabelling), P and N maps collected and merged, perimeters around N-rich regions mapped (as in Fig. 3C), and nucleoplasmic structures with diameters and P:N ratios typical of active transcription sites (i.e. >40 nm and 0.38±0.1) selected; three examples are shown. These sites by definition have similar diameters and P:N ratios to their labelled counterparts in permeabilised cells; they also have similar textures. Scale bars: 1 µm (A,B); 100 nm (C-H).

 

Figure 5
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  		Fig. 5. Site morphology. Merges were prepared as in Fig. 3D (A-D,F) and Fig. 4C (E). Arrows indicate transcription sites marked by gold particles. (A) Site marked by two gold particles surrounded by space free of P and N. (B) Site marked by particles surrounded by chromatin (arrowhead marks RNPs and/or nucleosomes). (C) Three sites marked by particles lying close to each other. (D) Site marked by particles lying near other gold-free structures (arrowheads) that have dimensions and P:N ratios typical of sites. (E) Site containing many P-rich granules (arrowhead) smaller than nucleosomes. This site was selected by correlative microscopy because there are no obscuring gold particles (but similar granules are seen by immunogold labelling). Inset, P map. (F) Site lying near IGC (arrowhead). Scale bars: 100 nm (A,B,D,E); 50 nm (C,F).

 

Figure 6
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  		Fig. 6. Cartoon illustrating a section through a site. An active polymerase (attached to a core) reels in its template (blue arrow) as it extrudes its transcript; only one of ~eight polymerising complexes on the surface is shown. The upper face of the section illustrates how this structure appears in an ESI merge. The N-rich core appears green, except for several (yellow) granules that each contain ~70 P atoms; nucleosomes and RNPs (both yellow) are found on/near the surface. Sectioning often removes one or other pole with any associated templates/transcripts, while leaving those tethered to equators. When viewed from above, most RNA is then seen associated with equators, with little apparently lying within cores (see Materials and Methods for quantitative analysis).

 

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