Higher-order epistasis within Pol II trigger loop haplotypes.

RNA polymerase II (Pol II) has a highly conserved domain, the trigger loop (TL), that controls transcription fidelity and speed. We previously probed pairwise genetic interactions between residues within and surrounding the TL and identified widespread incompatibility between TLs of different species when placed in the Saccharomyces cerevisiae Pol II context, indicating epistasis between the TL and its surrounding context. We sought to understand the nature of this incompatibility and probe higher order epistasis internal to the TL. We have employed deep mutational scanning with selected natural TL variants ("haplotypes"), and all possible intermediate substitution combinations between them and the yeast Pol II TL. We identified both positive and negative higher-order residue interactions within example TL haplotypes. Intricate higher-order epistasis formed by TL residues was sometimes only apparent from analysis of intermediate genotypes, emphasizing complexity of epistatic interactions. Furthermore, we distinguished TL substitutions with distinct classes of epistatic patterns, suggesting specific TL residues that potentially influence TL evolution. Our examples of complex residue interactions suggest possible pathways for epistasis to facilitate Pol II evolution.