Fig. 1: Schematics of the Pol II active site interaction landscape.

a The Pol II active site is embedded in the center of a 12-subunit complex (left panel). Pol II functions are supported by distinct TL conformational states. An open TL (PDB: 5C4X) and closed TL (PDB: 2E2H) conformations are shown in the middle panel. GOF mutations have been identified in the TL and its proximal domains (right panel), suggesting TL mobility and function may be impacted by adjacent residues. b Examples of inter-residue genetic interactions. WT residues are shown in circles with a number indicating residue position in Rpb1. Mutant substitutions are shown in colored circles, with color representing mutant class. Colored lines between mutant substitutions represent types of genetic interactions. c Overview of experimental approach. We synthesized 10 libraries of TL variants represented by colored stars. Libraries were transformed into WT or mutated yeast strains. A selection assay was subsequently performed by scraping and replating the transformants onto different media for phenotyping. DNA was extracted from yeast from all conditions, and then went through TL region amplification and Illumina sequencing. Read counts for variants on general conditions were used to determine growth fitness, while read counts on other conditions were used to determine the phenotypic fitness landscape. d Overview of analytical approach for determining interaction landscape. Mutant conditional growth fitnesses were calculated using allele frequencies under selective growth conditions and subjected to two logistic regression models for classification/prediction of catalytic defects. Double mutant interactions were computed using growth fitness. Classification allowed epistatic interactions to be deduced from double mutant growth fitness (see “Methods”).