Transcription activation, a key step in gene control, is the readout of many signaling pathways controlling cell growth, development and stress response and defects in activation cause many human diseases and syndromes. Activation typically results from factors containing activation domains (ADs) binding to coactivator complexes containing activator binding domains (ABDs). Most known ADs are unstructured in the absence of a binding partner and many interact with multiple and structurally-unrelated ABDs. The mechanisms used by ADs to interact with coactivators, the specificity of these interactions, and mechanisms of coactivator cooperativity are critical unanswered questions for understanding the molecular basis of gene regulation. The long-term goal of this project is to determine mechanisms used by gene-specific activators and coactivators to regulate RNA polymerase (Pol) II transcription. The objectives of this proposal are to determine: mechanisms used by ADs to recognize their coactivator targets, cooperative mechanisms used by the coactivators Mediator and TFIID, and what constitutes a functional AD and ABD. This work will utilize an interdisciplinary combination of biochemical, structural, molecular, and computational approaches to examine activation in S. cerevisiae. These mechanisms are likely to be conserved, since nearly all activators tested function in both yeast and mammalian cells. To understand these mechanisms, we will build upon several breakthrough concepts and methods developed in the past grant period. We will use protein crosslinking and mass spectrometry, combined with state-of-the-art NMR to examine mechanisms used by broadly-acting ADs when binding their coactivator targets - in both large physiologically relevant complexes and at the atomic level. We will examine cooperativity between Mediator and TFIID, two conserved and widely used transcription coactivators. Finally, we will use a combination of molecular, structural, and computational approaches to delineate what comprises AD function. These new approaches will reveal mechanisms of activator binding and specificity, mechanisms of coactivator cooperation, and find rules that explain a large class of broadly acting ADs. Our proposed research is significant because it will reveal new molecular recognition mechanisms that are important for understanding transcriptional regulation as well as a wide variety of interactions involving inherently disordered proteins that function in many biological systems.

Public Health Relevance

Regulation of transcription by activation of gene expression is a key mechanism for control of cell growth, differentiation, and development. Defects in activation directly contribute to many human illnesses. Understanding the mechanisms of activation and the molecular recognition properties of inherently disordered proteins will form the basis for understanding molecular defects in transcription disorders leading to many types of cancer, heart disease, neurological disorders, and birth defects. It will also provide the molecular basis for identifying new avenues of potential interventions and therapies to target specific protein-protein interactions in activator-coactivator and coactivator-coactivator complexes that are the readout of many important signaling pathways.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Sledjeski, Darren D
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Fred Hutchinson Cancer Research Center
United States
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Grünberg, Sebastian; Henikoff, Steven; Hahn, Steven et al. (2016) Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters. EMBO J 35:2435-2446
Han, Yan; Luo, Jie; Ranish, Jeffrey et al. (2014) Architecture of the Saccharomyces cerevisiae SAGA transcription coactivator complex. EMBO J 33:2534-46
Hahn, Steven (2014) Ellis Englesberg and the discovery of positive control in gene regulation. Genetics 198:455-60
Warfield, Linda; Tuttle, Lisa M; Pacheco, Derek et al. (2014) A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface. Proc Natl Acad Sci U S A 111:E3506-13
Grünberg, Sebastian; Hahn, Steven (2013) Structural insights into transcription initiation by RNA polymerase II. Trends Biochem Sci 38:603-11
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Knutson, Bruce A (2010) Insights into the domain and repeat architecture of target of rapamycin. J Struct Biol 170:354-63

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