PROJECT SUMMARY Advances in eukaryotic gene expression have provided a detailed and 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, revealing a transcriptionally poised chromatin architecture and preinitiation complex at gene promoters and enhancers. Despite these advances, major challenges remain in the integration of chromatin remodeling and modifying events with the complex orchestration of transcription enzymology that were traditionally defined on naked DNA templates, and 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 developing an in vitro RNA Polymerase II (Pol II) transcription system based on purified yeast minichromosomes that carry the natural set of chromatin architectures and modifications and is responsive to transcription activators. With this minichromosome platform, we aim to introduce defined alterations in chromatin and biochemically manipulate specific components of the transcription apparatus to elucidate causal and temporal relationships. Extension of the analysis to the single-molecule-level to circumvent the confounding asynchrony of ensemble reactions will allow detection of short-lived reaction intermediates, and definition of the temporal order of intermediate events. Thus, this approach allows a direct test of the hypothesis that the kinetics of the multi-step transcription process is regulated by chromatin-based mechanisms, answering questions beyond the reach of current methods and synergistic with live-cell imaging of transcription and chromatin remodeling.