The overall objective of this proposed project is to investigate the relationship between eukaryotic transcriptional regulation and chromatin structure, using Saccharomyces cerevisiae as a model organism for these studies. The research design focuses on yeast-purified multisubunit complexes that are capable of using nucleosomes as a substrate for acetylation. Two complexes that have been purified and partially characterized were shown to contain the transcriptional adaptors GCN5 (a histone acetyltransferase) and ADA2. One of these complexes, SAGA, also contains several transcriptionally relevant SPT proteins. For the proposed studies, the SAGA complex will be tested in vitro for interaction with the TATA-binding protein (TBP) and activators, two crucial components in activated transcription. The structural and functional relationship between the adaptor and SPT subunits of SAGA will also be investigated by interaction and acetylation studies. In addition, the roles of SAGA components in transcription itself will be examined by preparing complexes from adaptor and spt mutants and employing them in an in vitro activated transcription system with a nucleosomal DNA template. These studies are intended to address fundamental mechanisms of the regulation of eukaryotic gene expression, which likely have relevance to complex human disease processes such as cancer. Since transcriptional regulation has long been recognized as important in these areas, and chromatin is increasingly discovered to have a significant role therein, the proposed project may suggest novel approaches to disease treatment by providing a better understanding of regulatory pathways.
