Chromatin Remodeling Complexes: RSC
Chromatin remodeling complexes (remodelers) are large, multisubunit macromolecular assemblies that utilize ATP hydrolysis to alter the structure and positioning of nucleosomes. The mechanisms proposed for remodeler action on nucleosomes are diverse, and require structural evaluation and insights. Previous reconstructions of remodelers using electron microscopy revealed interesting features, but also significant discrepancies, prompting new approaches. We have devised a novel Orthogonal Tilt Reconstruction method for single particle 3D reconstruction, which is well suited to heterogeneous samples, and used it to provide a new reconstruction of the yeast RSC complex. We were able to reveal two interesting features: first, we observed a deep central cavity within RSC, displaying a remarkable surface complementarity for the nucleosome. Second, we were able to visualize two distinct RSC conformers, revealing a major conformational change in a large protein 'arm' which may shift to further envelop the nucleosome. Our structures provide both support and challenges for present models of nucleosome sliding. The shape of the deep cavity and its remarkable fit to the nucleosome sets limits on the size of DNA waves/loops that can be propagated along the entire length of the nucleosome and suggests that very small loops are likely the primary intermediate for the sliding reaction.
Chromatin Remodeling Complexes - PBAF
The SWI/SNF family of chromatin remodeling complexes uses ATP hydrolysis to modify the structure of nucleosomes in order to allow access of DNA-interacting proteins to their target sequences. Two closely related human chromatin remodeling factors, SWI/SNF-A and SWI/SNF-B (or P-BAF) were identified and purified in the Tjian lab (Lemon et al., 2001). We have determined the structure of the human PBAF by single particle analysis of negatively stained samples (Leschziner et al., Structure 2005). The complex contains a larger density to which two smaller knobs are attached, surrounding a central cavity. One of these knobs appears remarkably flexible. Using two-dimensional image analysis we have mapped the nucleosome-binding site to the central cavity in contact with the flexible knob. Thus, the flexibility of this region might be biologically relevant during the remodeling cycle. We are now studying a number of closely related human, fly, and yeast complexes in collaboration with the labs of Robert Tjian and Brad Cairns (University of Utah), with the goal of understanding the general mechanism of nucleosome remodeling and the specific properties of each complex.