Widespread epistasis shapes RNA polymerase II active site function and evolution.

Multi-subunit RNA Polymerases are responsible for transcription in all kingdoms of life. These enzymes rely on dynamic, highly conserved active site domains such as the so-called "trigger loop" to accomplish steps in the transcription cycle. Mutations in the RNA polymerase II trigger loop confer a spectrum of biochemical and genetic phenotypes that suggest two main classes, which decrease or increase catalysis or other nucleotide addition cycle events. The RNA polymerase II active site relies on networks of residue interactions to function, and mutations likely perturb these networks in ways that may alter mechanisms. Here, we take a structural genetics approach to reveal residue interactions within and surrounding the RNA polymerase II trigger loop - determining its "interaction landscape" - by deep mutational scanning in Saccharomyces cerevisiae RNA polymerase II. This analysis reveals connections between trigger loop residues and surrounding domains, demonstrating that trigger loop function is tightly coupled to its specific enzyme context.