PROJECT SUMMARY Transcription in eukaryotes requires RNA polymerase II (Pol II) to faithfully synthesize RNA relative to DNA sequence. Pol II must also successfully negotiate obstacles within chromatin templates and coordinate cotranscriptional events. Disruption of any of these processes may alter gene expression outcomes and derange cellular growth or physiology. Pol II, like all multisubunit cellular RNA polymerases (msRNAPs), contains an essential active site domain – the trigger loop (TL) – that participates in all basic nucleotide addition cycle (NAC) functions. Changes to Pol II catalytic activity have the power to alter every phase of transcription: initiation, elongation, termination, and cotranscriptional RNA processing. It is therefore likely that evolution of Pol II function has involved complex trade-offs between the rate, efficiency and fidelity of these processes. Structural, biochemical and genetic studies alike on Pol II from Saccharomyces cerevisiae have enabled critical insight into eukaryotic transcription. The high conservation of the S. cerevisiae transcription machinery and its amenability to a wide-range of experimental approaches make yeast uniquely suited to decipher gene expression mechanisms. We leverage novel approaches from high-throughput quantitative phenotyping, structural analysis of unique Pol II variants, and determination of Pol II-natural product interactions to test existing, and generate new, models for Pol II function. Misregulation of gene expression is a major contributor to human disease. Basic research into transcription mechanisms enables the defects in transcription found in disease states to be better understood.