Project Summary/Abstract The essential multi-subunit RNA polymerases are regulated at each stage of the transcription cycle to control gene expression in each Domain. In many cases the rate limiting step of gene expression is during transcription elongation, but major knowledge gaps remain in our understanding of post-initiation regulation of transcription. Our studies directly address outstanding questions of transcription regulation in archaeal and eukaryotic cells. Our overarching goals are to establish molecular mechanisms that regulate post-initiation activities of RNAP, and establish the regulation imposed by histone-based chromatin on gene expression. How does altering the chromatin-landscape alter gene expression? How, in molecular detail, do factors that modify the activities of RNA polymerase accelerate transcription on histone-bound DNA? How can the otherwise extremely stable transcription elongation complex be disrupted to terminate transcription accurately? These complex challenges demand continued attention to define the foundational mechanisms underlying gene expression and the aberrant gene expression associated with disease states and cancer. We defined that archaeal transcription and chromatin systems are closely related, yet minimal versions of the component complex eukaryotic transcription systems. We have described the complete archaeal transcription cycle and defined three mechanisms that control transcription termination decisions, each of which reveals similarities with bacterial and eukaryotic termination mechanisms and demands continued experimentation to delineate conserved mechanisms to disrupt the transcription elongation complex. Our understanding of how chromatin structure regulates gene expression is also incomplete. We will leverage the simplicity of single-histone chromatin formed with native histones and histone-variants to establish the regulation imposed by extended chromatin structures on a genome-wide level. We will also describe the molecular activities of conserved archaeal-eukaryotic transcription factors that modify RNA polymerase and accelerate transcription through histone-bound DNA.