Project Summary Advances in eukaryotic gene expression have provided a comprehensive list of transcription-related proteins, their biochemical activities and structure-function relationships, and revealed the importance of histone modifications and nucleosome remodeling enzymes that cooperate with sequence-specific DNA binding transcription factors, resulting in a transcriptionally poised chromatin architecture at gene promoters and enhancers. However, major challenges remain in the lack of knowledge of the timescales and kinetics by which epigenetic and transcription proteins operate on chromatin substrates. This proposal will address these challenges by focusing on quantitative kinetics of chromatin remodeling and transcription, using state-of-the-art single-molecule imaging techniques applied to living cells and immobilized chromatin templates in vitro, combined with traditional biochemistry and yeast molecular genetics. Anticipated findings are the identification of key reaction intermediates, order of events, and rate-limiting steps that will dramatically advance basic, mechanistic understanding of transcription on native chromatin with high impact on other areas of eukaryotic DNA metabolism.