The conserved trigger loop (TL) lies in the RNA polymerase II (Pol II) active site, multi-tasking in substrate selection, catalysis, and translocation. To dissect TL function at individual-residue resolution, we quantitatively phenotyped nearly all possible TL single substituted mutants en masse in Saccharomyces cerevisiae. Three major mutants classes were revealed by clustering of growth phenotypes linked to transcription defects or various stresses. TL phenotypic classes have distinct distributions within the TL, and distinguish previously characterized catalytically fast and slow mutants. Substitutions in residues A1076, M1079, T1080, and L1101 support the proposed function of an intra-TL hydrophobic pocket in stabilizing the open TL conformation. Disruption of this pocket confers phenotypes consistent with hyper-activation of Pol II. In addition, allele-specific suppression/exacerbation between newly characterized TL mutants and mutants in the TL adjacent domains is consistent with the contribution of the TL adjacent funnel and bridge helices to TL dynamics. Our phenotyping platform, which is readily applicable to other yeast proteins, enables incorporation of structural and phenotypic data to dissect the Pol II active site with high functional resolution.