The p53 transcription factor, which is the focus of this work, is a potent tumor suppressor that works by activating expression of genes that control key cellular processes such as growth arrest and apoptosis. In order to activate its target genes, p53 interacts with a protein complex known as Mediator. Mediator helps regulate expression of nearly all protein-coding genes;thus, it lies at the heart of transcriptional regulation. Unlike most transcription factors, p53 interacts with Mediator via 2 different domains. Notably, using electron microscopy (EM) and single-particle reconstruction techniques, we have shown that each different p53 domain induces a dramatically different Mediator conformational state upon binding to the complex. The goal of this work is to clearly define the mechanistic role of each p53-Mediator interaction and how they may work together to regulate p53 activity. Experiments described in Aim 1 will use a reconstituted, human in vitro transcription system to examine how p53-induced structural changes affect the biochemical function of Mediator.
In Aim 2 we will use immobilized template assays to explore whether p53-Mediator structural changes alter the assembly, composition, or stability of the transcriptional machinery (a.k.a. the Pre- Initiation Complex ).
In Aim 3, we will combine cryo-EM studies with p53 crystal structure docking to reveal how different domains within the p53 tetramer work together to orchestrate such dramatic structural changes in Mediator. Taken together, these biochemical and structural studies will provide much-needed insight into the molecular mechanisms by which Mediator and p53 work together to control expression of key tumor suppressor genes.p53 is one of the most commonly mutated genes in human cancer. In order to properly function in preventing tumor formation, p53 must interact with a protein complex known as Mediator. Our work will define the fundamental molecular mechanisms by which p53 and Mediator work together to control expression of key anti-cancer genes. This will enhance our understanding of how p53 works to safeguard human cells against cancer;moreover, we anticipate that the information accumulated by our efforts will identify new strategies for controlling the anti-cancer activity of p53.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA127364-05
Application #
8099570
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Knowlton, John R
Project Start
2007-09-21
Project End
2012-07-31
Budget Start
2011-08-01
Budget End
2012-07-31
Support Year
5
Fiscal Year
2011
Total Cost
$220,332
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Bunch, Heeyoun; Zheng, Xiaofeng; Burkholder, Adam et al. (2014) TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release. Nat Struct Mol Biol 21:876-83
Bancerek, Joanna; Poss, Zachary C; Steinparzer, Iris et al. (2013) CDK8 kinase phosphorylates transcription factor STAT1 to selectively regulate the interferon response. Immunity 38:250-62
Davis, Michael A; Larimore, Elizabeth A; Fissel, Brian M et al. (2013) The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator. Genes Dev 27:151-6
Poss, Zachary C; Ebmeier, Christopher C; Taatjes, Dylan J (2013) The Mediator complex and transcription regulation. Crit Rev Biochem Mol Biol 48:575-608
He, Yuan; Fang, Jie; Taatjes, Dylan J et al. (2013) Structural visualization of key steps in human transcription initiation. Nature 495:481-6
Lai, Fan; Orom, Ulf A; Cesaroni, Matteo et al. (2013) Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494:497-501
Lin, Shih-Chieh; Karoly, Edward D; Taatjes, Dylan J (2013) The human ?Np53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function. Aging Cell 12:863-72
Schwartz, Jacob C; Ebmeier, Christopher C; Podell, Elaine R et al. (2012) FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2. Genes Dev 26:2690-5
Bernecky, Carrie; Taatjes, Dylan J (2012) Activator-mediator binding stabilizes RNA polymerase II orientation within the human mediator-RNA polymerase II-TFIIF assembly. J Mol Biol 417:387-94
Taylor, Derek J; Podell, Elaine R; Taatjes, Dylan J et al. (2011) Multiple POT1-TPP1 proteins coat and compact long telomeric single-stranded DNA. J Mol Biol 410:10-7

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