The experiments in this proposal will characterize the chromatin structure of the genes and promoter elements of the 5S and 35S ribosomal genes in yeast. Nuclease and chemical cleavage will characterize the primary level of chromatin structure across the approximately 5 kb region which includes the genes and their promotors, by indirect end label analysis, applying both low resolution (nucleosome patterns and locations, hypersensitive regions) and high resolution (nuclear footprinting of DNA/protein interactions) approaches. A technique allowing similar analysis in vivo will be developed and applied. Several interesting regions including the defined promoters and terminators for the ribosomal genes will be of particular interest. The role of aspects associated with the higher orders of chromatin structure (matrix attachment, supercoiling) in the function of these elements will also be assessed. The structural data will be used as an assay to isolate trans-acting factors involved in the proper function of the gene control elements for these genes. We will utilize a unique system which allows one to probe a single riboxomal gene in the midst of the approximately 100 other copies in the yeast cell. By determining the unambiguous structure of these elements in various expression states, we should learn more about how these elements function in gene control. The combined structural/functional analysis of this well defined system will give novel insights on the relation of chromatin structure to control of gene expression in eukaryotes. An understanding of the bases for the control of gene expression is absolutely essential in understanding and solving a number of outstanding health problems, for example, cancer, aging and processes like abnormal development, which affect humans and other multicellular organisms. However, the latter systems are very complex. If one can gain an understanding of some of the bases for control of gene expression in a system like yeast, which has fewer complexities and is amenable to very favorable manipulations, this knowledge will be useful in helping to suggest approaches which can lead to understanding in the more complex systems.
