Project Summary/Abstract The human genome measures nearly 2 meters in length and must be compacted to fit in the nucleus, which measures only a few microns in diameter. In addition to compaction, the DNA must be organized to maintain genome integrity but also remain accessible to the molecular machines that read the genome. How the genome is organized directly influences which genes are expressed in a particular cell. Decoupling of genome organization and gene expression has profound consequences for cells, leading to cancer, intellectual disability, and developmental delay. It is mechanistically unclear how genome organization and the first step of gene expression, transcription, are physically coupled. Determining how both processes are linked is critical for understanding how cell type function and specificity are achieved. The first level of eukaryotic genome compaction and organization is mediated by nucleosomes that are formed by wrapping DNA around histone proteins. Here I will investigate how DNA sequence and nucleosomes impact gene expression in promoter proximal regions of human genes. I will mechanistically decipher how the processes of transcription and genome compaction are intertwined by reconstituting transcription reactions on chromatin in vitro and observing them using (1) a novel high throughput biochemical assay and (2) time-resolved cryo-electron microscopy (EM). In the first part of this proposal, we will develop a high throughput, single molecule transcription assay using reconstituted transcription complexes on thousands of DNA sequences. We will assess how DNA sequence, chromatin state, and transcription factors directly regulate transcriptional activity during early transcription elongation. This assay will overcome major hurdles in the field by revealing directly how nucleosomes and protein factors affect transcription behavior on an unprecedented number of DNA sequence contexts. In the second part of this proposal, we will define how chromatin and DNA sequence influence transcription dynamics by capturing structural snapshots of transcription complexes as they transcribe through nucleosomes. We will use time-resolved cryo-EM to directly assess which states transcription complexes adopt during early transcription and identify stable online and labile offline states. Together, this ambitious proposal will address important questions regarding how nucleosomes and DNA sequence are used to directly influence transcriptional output. The methods and data acquired in this proposal will help fulfill the long-term vision of my lab to fully reconstitute mammalian transcription to determine how cell fate is regulated by the coupling of genome organization and gene expression and how disease mutations can perturb this coupling.