In the nucleus, DNA is tightly packaged into a high order structure known as chromatin, and as such is inaccessible to the transcription machinery. Therefore, controling access to DNA by altering the structure of chromatin constitues an important regulation mechanism. A large number of macromolecular complexes are responsible for chemical and physical modifications to chromatin structure that render chromosomal DNA accessible to the transcription machinery. We are pursuing structural characterization of RSC, an essential ATP-dependent chromatin remodeling complex that induces physical changes in chromatin structure. We are also studying the structure of NuA4, a histone acetyltransferase (HAT) complex responsible for chemical changes to the histones that alter their interaction with DNA.

RSC particles preserved in stain were imaged at two different angles. Reference-free alignment and multivariate statistical analysis were used to classify particle images into homogeneous groups. A 3-D structure of RSC was calculated using the random conical tilt reconstruction method. Two different views of the structure illustrate its most prominent feature: a large central cavity that matches closely the size and shape of a nucleosome core particle.

Whereas the shape of structural subdomains remains constant, their relative position and orientation can vary significantly. Different conformational states of the RSC complex are likely to display different levels of accessibility to the central cavity.

Incubation of RSC with nucleosomes leads to formation of a RSC-nucleosome complex that can be characterized biochemically. RSC and related remodeling complexes expose DNA to attack by nucleases. However, upon binding of the nuclesome by RSC, the rate of double-strand celavage by nuclease, and of single-strand cleavage by DNase I, are reduced, independent of ATP. This suggests that RSC envelopes the nucleosome. In fact, an EM reconstruction of the RSC-nucleosome complex indicates the presence of density in the central cavity of the RSC structure. However, this density is relatively small compared to the expected size of a nucleosome core particle, perhaps due to partial dissociation of the RSC-nucleosome complex under EM sample preparation conditions.

Structural analysis of the NuA4 HAT complex is currently underway. Structures of the RSC-nucleosome and NuA4-nucleosome complexes are also being pursued. Antibody and metal-cluster tagging, as well as structural analysis of deletion mutants, are being pursued to determine the location of different subunits in the RSC and NuA4 complexes.