Abstract: Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present a high-resolution map of Pol II occupancy across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for noncoding RNA genes. The wild-type pattern of Pol II distribution provides unexpected insights into the mechanisms by which genes are repressed or silenced. Remarkably, a single amino acid substitution that compromises Sen1 function causes profound changes in Pol II distribution over both noncoding and protein-coding genes, establishing an important function of Sen1 in the regulation of transcription. Given the strong similarity of the yeast and human Sen1 proteins, our results suggest that progressive neurological disorders caused by substitutions in the human Sen1 homolog Senataxin may be due to misregulation of transcription.