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. At the heart of these msRNAPs is an ultra-conserved active site domain, the trigger loop (TL), coordinating transcription speed and fidelity by critical conformational changes impacting multiple steps in substrate selection, catalysis, and translocation. Previous studies have observed several different types of genetic interactions between eukaryotic RNA polymerase II (Pol II) TL residues, suggesting that the TL’s function is shaped by functional interactions of residues within and around the TL. The extent of these interaction networks and how they control msRNAP function and evolution remain to be determined. Here we have dissected the Pol II TL interaction landscape by deep mutational scanning in Saccharomyces cerevisiae Pol II. Through analysis of over 15000 alleles, representing all single mutants, a rationally designed subset of double mutants, and evolutionarily observed TL haplotypes, we identify interaction networks controlling TL function. Substituting residues creates allele-specific networks and propagates epistatic effects across the Pol II active site. Furthermore, the interaction landscape further distinguishes alleles with similar growth phenotypes, suggesting increased resolution over the previously reported single mutant phenotypic landscape. Finally, co-evolutionary analyses reveal groups of co-evolving residues across Pol II converge onto the active site, where evolutionary constraints interface with pervasive epistasis. Our studies provide a powerful system to understand the plasticity of RNA polymerase mechanism and evolution, and provide the first example of pervasive epistatic landscape in a highly conserved and constrained domain within an essential enzyme.