Functional and structural studies of the transition from transcription initiation to elongation

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The goal of this proposal to determine the mechanisms of pol II transitions between initiation, elongation, and re-initiation by pol II, which we will accomplish through structural and biochemical means using the powerful Saccharomyces cerevisiae model system. The in vitro transcription system we have developed for structural analysis is based on stoichiometrically assembled, highly active and homogeneous components comprising over 30 polypeptides, including RNA polymerase II (polII) and the general transcription factors (GTFs). Critically, we have recently succeeded in optimizing this in vitro reconstituted transcription system such that it achieves melting of double-stranded promoter DNA, and initiation of RNA synthesis de novo with ~100% efficiency. We will leverage this system to isolate and dissect, both structurally and biochemically a series of pol II complexes representing a range of states, from initiation, to early elongation, to complexes that might function as scaffolds for reinitiation. We will achieve these aims through Cryo-EM, cross-linking and mass spectrometry (XL-MS), and quantitative XL- MS.