1. The Nature of the Nucleosomal Barrier to Transcription
Jan Bednar, Vasily M Studitsky, Sergei A Grigoryev, Gary Felsenfeld, Christopher L Woodcock 
Molecular Cell 
Volume 4, Issue 3, Pages (September 1999)
DOI: /S (00)

2. Figure 1 Structure and Characterization of the 227 bp Templates
(A) Design of the pD70 and pD89 templates. Positions of nucleosomes on the templates are indicated by ovals; shaded box indicates the region of strong nucleosome-specific pausing (Studitsky et al. 1995). Positions of arrested complexes at the end of the −C tracks are indicated by vertical arrows. Promoter for SP6 RNA polymerase and position of unique site for HaeII restriction enzyme are indicated.
(B) Arrest of SP6 RNA polymerase at the unique positions on pD89 and pD bp DNA templates. pD89 or pD bp DNA fragments were incubated with SP6 RNA polymerase and 0.5 mM of ATP, GTP, and 32P-UTP in the absence or in the presence of CTP (− and +, correspondingly). RNA was resolved in a sequencing gel. Positions of the 70 nt, 89 nt, and run-off (194 nt) transcripts are indicated.
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2
Analysis of the pD70 and pD89 Nucleosomal Templates Using the Restriction Enzyme Sensitivity Assay
(A) Schematic diagram of expected nucleosome positioning and sensitivity to restriction enzymes of the pD70 and pD89 templates before and after transcription. Before transcription SspI and AseI sites were expected to be sensitive, and PacI, BglII, and HaeII resistant to the restriction enzymes. After transcription, SspI, AseI, PacI, and BglII sites were expected to be resistant, and HaeII sensitive to the restriction enzymes.
(B) Sensitivity of the pD89 nucleosomal template to restriction enzymes. pD bp nucleosomal templates end-labeled at the NcoI site were incubated with RNA polymerase in the presence of ATP only (−Transcription), in the presence of ATP, GTP, and UTP (−CTP), or in the presence of all NTPs (+Transcription), and then incubated in the presence of one of the indicated restriction enzymes (S, SspI; A, AseI; P, PacI; B, BglII; H, HaeII). Since transcription continues during restriction digestion, efficiency of template utilization is very high. The resulting complexes were subjected to nucleoprotein gel electrophoresis. Positions of nucleosomes and free templates in the gel are indicated on the left. Note that mobility of the arrested (−CTP) complex (without digestion with restriction enzymes) is similar to mobility of nucleosomes before transcription; it was found that SP6 RNA polymerase dissociates from the templates during the electrophoresis and therefore lower mobility complexes containing the elongation complex are not detected in the gel. Black circles indicate positions of PacI-digested nucleosomes in the gel. M, end-labeled MspI digest of pBR322.
(C) Sensitivity of the pD70 nucleosomal template to restriction enzymes. Brackets indicate positions of BglII-digested nucleosomes in the gel; heterogeneous mobility of BglII-digested nucleosomes is likely to occur due to partial dissociation of DNA from the histone octamer during electrophoresis.
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3
DNase I Footprinting of the pD70 and pD89 Nucleosomal Templates Containing Arrested Elongation Complexes
(A) DNase I footprinting of the nucleosomal templates. Free or nucleosomal pD templates end-labeled at NcoI site (nontemplate DNA strand) were incubated without RNA polymerase (−RP), or with RNA polymerase in the presence of ATP only (−Tr.), in the presence of ATP, GTP, and UTP (−C), or in the presence of all NTPs (+Tr.), and incubated in the presence of DNase I. DNA was resolved in a sequencing gel. M, positions of lanes with DNA sequencing markers. The second lane from the left in each panel is undigested DNA. Numbers on the right are distances (nt) from the SacI end of the templates. Positions of polymerase footprints in the arrested complexes are indicated with brackets. Intranucleosomal sites that become more accessible in the arrested complexes are indicated. Position of the nucleosome before transcription is indicated on the right. Bands generated due to predigestion of nucleosomes with HaeII during isolation are labeled with asterisks.
(B) Quantitation of DNase I footprints. The extent of protection of the 1–80 region from DNase I (pD89 nucleosomal templates) before (−Tr., solid line) and after transcription (+Tr., dashed line) was quantitated using a PhosphorImager. The amount of radioactivity in each lane was corrected for loading, and overall extents of digestion were comparable. Protection of the 10–80 region is about 50% higher after transcription. Similar data were obtained using the pD70 template.
(C) Schematic summary of sensitivity of the pD70 and pD89 nucleosomal templates to different probes. Positions of nucleosomes before (−NTP) and after (+NTP) transcription are indicated by large ovals. In the arrested complexes (−CTP), the nucleosomes are positioned as before transcription. Positions of relevant sites for the restriction enzymes are indicated: “−,” “+,” and “+/−” indicate resistant, accessible, and partially accessible sites, respectively. Sites of increased sensitivities to DNase I are indicated by arrows. Positions of footprints of SP6 RNA polymerase on DNA are indicated by black boxes.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4 ECM Visualization of Transcripts and Nucleosomes
(a) ECM images and (b) interpretive representations of naked pD89 DNA with arrested SP6 RNA polymerase. Arrows indicate the location of the polymerase–RNA complex at the arrest site. (c) 3D reconstruction of the particle marked (*), presented as a stereo pair. Bar, 10 nm. (d) ECM images of pD70 mononucleosomes in transcription buffer but without polymerase. One-tailed mononucleosomes are the predominant conformation. Bar, 10 nm.
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5 Histograms of DNA Tail Length in One-Tailed Particles
The histograms were derived from 3D measurements on tilt pairs of ECM images. (Top) pD70 mononucleosomes in transcription buffer without polymerase. (Center) pD70 mononucleosomes with polymerase arrested. (Bottom) pD89 mononucleosomes with polymerase arrested.
Molecular Cell 1999 4, DOI: ( /S (00) )

7. Figure 6 ECM Images of Arrested and Transcribing Complexes
(a–l) pD89 particles with arrested polymerase. (a–f) One-tailed particles, some with polymerase–RNA complex (arrowheads) close to the octamer (designated CTI-I; a, e, and f), some with free DNA between polymerase and octamer (OTI complexes; b–d). (g–j) Two-tailed particles (designated CTI-II). Putative polymerase–RNA complexes appear as electron-dense regions at the periphery of the nucleosomes (arrowheads). (k and l) One-tailed particles in which the promoter-proximal DNA is considerably bent, a conformation that could lead to a transient looped intermediate (see text). Bar, 10 nm. At top is a 3D model of the complex shown in (a) viewed from two angles. The nucleosome is represented by a red disc, and the transcript by a black cylinder. (m–p) Gallery of images from running transcription experiments. After 10 s transcription, particles with a conformation similar to the CTI-I (m) and CTI-II complexes (n) are seen. In (o), the polymerase has evidently emerged from the nucleosome (based on the large size of the polymerase–RNA complex), and the promoter-proximal DNA is not yet fully wrapped in the octamer. After 30 s of reaction, transcription is either complete or the polymerase complex is seen close to the NcoI end of the DNA (p). Image interpretation, including identification of polymerase–RNA complexes is based on stereo viewing and 3D measurement of tilt pairs both from the original micrographs and CTF-processed images. CTF processing aids visualization of DNA trajectories but hinders discrimination of transcripts.
Molecular Cell 1999 4, DOI: ( /S (00) )

8. Figure 7
Proposed Sequence of Events during Transcription through a Nucleosome
The orientation of the octamer is fixed in all the drawings. (1) After RNA polymerase (RP) initiates transcription at the promoter (P) on the 227 bp template, it rapidly transcribes the first ∼25 bp of nucleosomal DNA (shown in dark contrast, boundary B), causing partial dissociation of DNA from the octamer (2). The DNA behind the RP binds transiently to the exposed surface of the octamer forming a loop (3). The advance of RP within this relatively small loop creates superhelical stress, which destabilizes the initial loop. This can lead to collapse of the loop and formation of a one-tailed intermediate (CTI-1) where the RP is in close proximity to the histone core (4). The RNA polymerase now interferes with the NcoI end of the DNA, eventually causing its displacement and the formation of a two-tailed paused transcriptional complex, CTI-II (5), in which the further movement of RP is inhibited due to direct contact of the polymerase with the histone octamer. The dynamic equilibrium of the system, together with the force exerted by the polymerase, results in its dissociation from the octamer, and the OTI configuration (2) is restored. This cycle of events may be repeated several times (not shown), as the polymerase progresses in ∼10 bp increments. Eventually, when the polymerase has penetrated ∼60 bp into the core, the tendency of the downstream portion of DNA to dissociate increases, giving rise to the conformation shown in (6) and completing the upstream transfer of the octamer. Transcription continues freely to the end of the template (7). With the exception of the short-living intermediate (marked with an asterisk), which was deduced from the biochemical experiments, all the intermediate conformations were seen in ECM images. Drawn roughly to scale.
Molecular Cell 1999 4, DOI: ( /S (00) )