phenotypic landscape of the RNA Polymerase II trigger loop 1 2

21 22 23 The active site of multicellular RNA polymerases have a " trigger loop " (TL) that 24 multitasks in substrate selection, catalysis, and translocation. To dissect the 25 Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we 26 quantitatively phenotyped nearly all TL single variants en masse. Three major mutant 27 classes, revealed by phenotypes linked to transcription defects or various stresses, have 28 distinct distributions among TL residues. We find that mutations disrupting an intra-TL 29 hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding 30 through destabilization of a catalytically inactive TL state, confer phenotypes consistent 31 with pocket disruption and increased catalysis. Furthermore, allele-specific genetic 32 interactions among TL and TL-proximal domain residues support the contribution of the 33 funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach 34 incorporates structural and phenotypic data for high-resolution dissection of transcription 35 mechanisms and their evolution, and is readily applicable to other essential yeast 36 proteins.