Project Summary. The goal of this project is to define the interactions between RNA polymerase II, the basal and regulatory transcription factors, and the chromatin template that lead to accurate transcription initiation and productive elongation. Using a combination of approaches in the yeast Saccharomyces cerevisiae model system, several fundamental aspects of gene expression will be studied. Specific Aim 1 will continue our studies of how transcription intersects with chromatin modifications. We will probe the function of the Set1/COMPASS complex, which methylates histone H3 at lysine 4 (H3K4). Methylation is co-transcriptional, and H3K4 trimethylation is strongest at the nucleosome nearest the transcription start site, while dimethylation predominates further downstream through the next few nucleosomes. This aim will explore how Set1/COMPASS is recruited to transcription complexes and how the gradient of methylation is created. In the previous funding period we successfully developed a pipeline for proteomic analysis of both initiation and elongation complexes using immobilized chromatin templates and quantitative mass spectrometry. Specific Aim 2 will exploit these systems to further define the events and factors that drive the transcription cycle forward. We also test a new model for how TFIID functions. In Specific Aim 3, our immobilized template transcription system will be combined with single-molecule TIRF microscopy to visualize transcription in real time. We will analyze the interaction kinetics between factors and the transcription template. Interesting findings in all these aims will be validated in vivo using the genetic, genomic, and molecular techniques our lab has developed over many years. The experiments in these three specific aims will significantly increase our understanding of the RNA polymerase II transcription reaction and its interactions with the chromatin template. Although the project uses a model system, mechanisms of transcription are highly conserved in eukaryotes and, based on past experience, the results are likely to be directly applicable to human gene expression. This fundamental knowledge is essential for understanding how mutations in transcription factors and histone modifying enzymes lead to diseases such as cancer and developmental defects.