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. 2002 Oct 15;99(21):13477-80.
doi: 10.1073/pnas.162504299. Epub 2002 Oct 4.

Structural analysis of the RSC chromatin-remodeling complex

Francisco J Asturias  1 , Wen-Hsiang Chung, Roger D Kornberg, Yahli Lorch
Affiliations

Structural analysis of the RSC chromatin-remodeling complex

Francisco J Asturias et al. Proc Natl Acad Sci U S A. .

Abstract

Electron microscopy of the RSC chromatin-remodeling complex reveals a ring of protein densities around a central cavity. The size and shape of the cavity correspond closely to those of a nucleosome. Results of nuclease protection analysis are consistent with nucleosome binding in the cavity. Such binding could explain the ability of RSC to expose nucleosomal DNA in the presence of ATP without loss of associated histones.

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Figures

Figure 1
Figure 1
Structure of RSC in projection. A total of 5,880 RSC particles preserved in uranyl acetate were computationally aligned and sorted into homogeneous classes (according to their conformation) using Hierarchical Ascendant Classification (16). A central area of lower density is apparent in several of the class averages shown (≈45% of particles). Most of the variation in RSC conformation is related to a domain forming the bottom part of the structure, which is either missing (≈35% of particles) or collapsed against the top of the structure (≈22% of particles).
Figure 2
Figure 2
The RSC chromatin-remodeling complex. (A) RSC consists of four modules that define a central cavity. Two views of the structure (front and back) are shown. Scale bar corresponds to 100 Å. (B) The most significant variation in RSC conformation was caused by the collapse (Upper) or absence (Lower) of a module that forms the lower part of the RSC structure.
Figure 3
Figure 3
Possible mode of RSC–nucleosome interaction. An x-ray structure of the nucleosome (20) was filtered to 25 Å and manually fitted in the central cavity of the RSC structure by using the program o (21). The close fit between the nucleosome and the RSC cavity is apparent in two different views (front and top). Scale bar corresponds to 100 Å.
Figure 4
Figure 4
Nuclease protection analysis of RSC–nucleosome complexes. (A) Micrococcal nuclease digestion. Nucleosomes (25 ng DNA) were treated with RSC (125 ng, ■ and ▴) or no RSC (●), and with ATP (0.5 mM, ▴) or no ATP (● and ■). Micrococcal nuclease was then added in the amounts indicated (u, units) for 5 min at 37°C, followed by 15 μl of 50 mM Tris⋅Cl, pH 8/20 mM EDTA/0.5 M NaCl/1% SDS. DNA was recovered by digestion with proteinase K and phenol-chloroform extraction, followed by electrophoresis in a 7% polyacrylamide gel in TBE buffer. The percentage of the DNA remaining in the band due to the full-length 154-bp fragment was determined with a PhosphorImager. (B) DNase I footprinting. Nucleosomes containing 3′-end-labeled DNA, or the DNA alone, were treated with RSC and ATP or not as indicated, followed by DNase I digestion and gel electrophoresis, all as described (6).
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