Eukaryotic cells deploy at least three multisubunit, DNA-dependent RNA polymerases to express the robust variety of RNAs required for cell survival and proliferation. In contrast to prokaryotic cells which express a single RNA polymerase, eukaryotic RNA polymerases have evolved into specialized cellular roles. RNA polymerase I (Pol I) synthesizes the majority of ribosomal RNA, Pol II synthesizes messenger RNA and most regulatory RNAs, whereas Pol III synthesizes transfer RNA and the 5S ribosomal RNA. While this "division of labor" among the nuclear Pols has been appreciated for many years, substantial gaps in our understanding of the functional divergence between the eukaryotic RNA polymerases remain. How do the enzymatic properties of these enzymes differ? How do these functional differences influence regulation of transcription by associated transcription factors? How do divergent enzymatic properties of the Pols impact their unique cellular roles? The answers to these questions are fundamentally important for understanding eukaryotic biology. It is well established that the rate of ribosome synthesis is proportional to the rates of cell growth and proliferation. As a consequence, several labs around the world seek novel inhibitors of ribosome synthesis as potential anticancer chemotherapy agents. Since transcription of the ribosomal DNA by Pol I is the first, rate-limiting step in ribosome synthesis, Pol I has emerged as a key target for the development these inhibitors. In order to selectively inhibit one RNA polymerase without affecting the others, it is crucial to understand the fundamental properties of the enzymes as well as the cellular mechanisms by which the enzymes are controlled. Thus, defining Pol I activity and its cellular roles has immediate translational value. By defining the landscape of eukaryotic transcription and the many cellular roles of Pol I, this project will reveal answers to fundamental questions in evolutionary biology while informing ongoing studies aimed at therapeutic targeting of ribosomal RNA synthesis. To reach these goals, our lab has developed a robust platform of experimental approaches including biochemical, biophysical, genetic, and genomic methodologies. We have pioneered the development of both the experimental and analytical strategies required to rigorously and thoroughly define the functional properties of RNA polymerase I. The overall goal of this project is to leverage these experimental strengths to impact our overall understanding of eukaryotic gene expression and to lay the foundation for ongoing and future studies aimed at therapeutic inhibition of Pol I.