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

Structurally conserved multi-subunit RNA Polymerases (msRNAPs) are responsible for cellular genome transcription in all kingdoms of life. At the heart of these RNA polymerases is an ultra- conserved active site domain, the trigger loop (TL). The TL participates in substrate selection, catalysis, and translocation of transcription elongation by switching between an open, catalytic- disfavoring state and a closed, catalytic-favoring state in the active site and is therefore central to the msRNAP nucleotide addition cycle. Previous studies have observed diverse genetic interactions between eukaryotic RNA polymerase II (Pol II) TL residues, supporting the idea that the TL’s function is shaped by functional interactions of residues within the TL and between the TL and its proximal domains. The nature and conservation of these intra-TL and inter-TL residue interaction networks, as well as how they control msRNAP function, remains to be determined. To identify the residue interactions that shape TL function and evolution, we have dissected the Pol II intra-TL and TL-Pol II residue interactions by deep mutational scanning in Saccharomyces cerevisiae Pol II. Through analysis of over 15000 alleles, representing single mutants, a subset of double mutants, and evolutionarily observed TL haplotypes, we identify interaction networks within the TL and between the TL and other Pol II domains. Substituting residues creates allele-specific networks and propagates epistatic effects across the Pol II active site. Our studies provide a powerful system to understand the plasticity of RNA polymerase mechanism and evolution.