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.
The transition from pol II initiation to elongation involves highly conserved and essential factors, and occurs at every gene. Although emerging evidence from recent genome- wide studies has revealed the transition is doubtless one of the most important control points in gene regulation, almost all the evidence comes from indirect studies of factor association with genes, with little insight into the mechanisms of factor exchange. The overall goal of this proposal is to define the timing of the transition from initiation to elongation and re-initiation and to determine the molecular basis for these transitions.
| Damodaren, Nivedita; Van Eeuwen, Trevor; Zamel, Joanna et al. (2017) Def1 interacts with TFIIH and modulates RNA polymerase II transcription. Proc Natl Acad Sci U S A 114:13230-13235 |
