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

Multi-subunit RNA Polymerases (msRNAPs) 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" (TL) to accomplish steps in the transcription cycle. Mutations in the RNA polymerase II (Pol II) TL confer a spectrum of biochemical and genetic phenotypes that suggest two main classes, which decrease or increase catalysis or other nucleotide addition cycle (NAC) events. The Pol II active site relies on networks of residue interactions to function and mutations likely perturb these networks in ways that may alter mechanisms. We have undertaken a structural genetics approach to reveal residue interactions within and surrounding the Pol II TL - determining its "interaction landscape" - by deep mutational scanning in Pol II. This analysis reveals connections between TL residues and surrounding domains, demonstrating that TL function is tightly coupled to its specific enzyme context.