Nucleosome structure has profound implications for DNA-templated processes and modulation of this structure is of critical importance for replication and transcription. Nucleosome structure must be loosened (reorganized) to facilitate polymerase passage and normal structure must be re-established in the wake of the elongating polymerase. Also, nucleosomes must be translated along DNA (remodeled) to allow access to promoter sequences and replication origins. Our goal is to understand the molecular basis for these activities, and our proposal is organized into three specific Aims, each of which focuses on a different protein or complex: Spt16-Pob3 (Aim 1) is a reorganizing complex that restructures nucleosomes to facilitate transcription initiation and passage of elongating RNA Pol II. It also performs an important (presumably equivalent) role in DNA replication.
Spt6 (Aim 2) performs a related role to Spt16-Pob3 by promoting reformation of normal nucleosome structure following passage of RNA Pol II. It also provides a link between the machineries for RNA transcription and processing.
RSC (Aim 3) is an ATP driven remodeling complex that functions in the expression of numerous genes by mediating nucleosomal translocation. Our approach is highly collaborative and broad. Because our goal is to arrive at a detailed molecular understanding we will determine relevant structures by x-ray crystallography (Hill, PI). Because we aim to understand processes in a biological context we are exploiting the strengths of yeast genetics (Formosa, co- I; and Cairns, collaborator). The link between structural and in vivo studies is provided by biochemical analysis, which is ongoing in all three labs. Further strength is added by our collaboration with experts in biophysical chemistry (David Myszka) and pathogen physiology and biochemistry (Simon Dove). We have made considerable progress toward the specific aims. In each case important structures have been determined and biochemical and genetic assays developed. Preliminary data indicate that considerable productivity will ensue and also lay the foundation for longer-term goals that carry more risk but also offer potential for high reward.
