This application addresses broad Challenge Area 06: Enabling Technologies and specific Challenge Topic 06-GM-101: Structural Analysis of Macromolecular Complexes. It focuses on the multiprotein Mediator complex, which has been implicated as a critical coactivator for transcription of essentially all RNA polymerase II-transcribed genes in eukaryotes. Through its ability to interact with both transcriptional regulators and the RNA polymerase machinery as well as through its multiple functional capabilities, Mediator is believed to be the main channel through which transcriptional signals are ultimately processed. Aberrant expression of several Mediator subunits has also been implicated in human diseases that include cancer, heart abnormalities, and neural degeneration. Despite their common evolutionary origins, the yeast and mammalian complexes have diverged considerably. Unlike the situation for the yeast Mediator, where genetic studies have highlighted important structure-function relationships and led the way for biochemical studies, technical issues have made it difficult to go beyond a gross description of the architecture of the 30-subunit mammalian complex and its function. Unaddressed issues relate to definition of the essential core of the complex and to the nature of other factors that impinge on its function under different conditions. Here we propose to undertake a systematic approach both to define the modular architecture of the core human complex (especially as it pertains to its essential functions) and to develop methods to purify the intact complex and its interacting proteins from a diverse range of biological sources. Towards these goals we aim (i) to recombinantly generate a minimal functional derivative of the modular Mediator complex;and (ii) to raise monoclonal antibodies against defined Mediator epitopes that will allow facile purification of the natural complex in association with interacting factors. Availability of these tools is thus expected to provide a jump-start to the next phase of studies in the human Mediator field.

Public Health Relevance

The multisubunit Mediator has emerged as one of the most critical factors involved in the precise transcription of protein-encoding genes, the aberrant expression of which is a cause of many human diseases, especially cancer. Although several individual subunits of the human Mediator have been directly implicated in diseases including colorectal cancer, heart abnormalities and neural degeneration, detailed studies of the human Mediator have faced significant technical challenges arising from its scarcity, extraordinary size and heterogeneity with respect to its composition. By directly addressing these challenges, this proposal aims to reveal new insights into the composition of the human Mediator, and, by generating important new antibody-based tools;
it aims to lay the foundation for anticipated future work into the molecular bases of many transcription-related diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
NIH Challenge Grants and Partnerships Program (RC1)
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Special Emphasis Panel (ZRG1-CB-N (58))
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Tompkins, Laurie
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Rockefeller University
Other Domestic Higher Education
New York
United States
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Cevher, Murat A; Shi, Yi; Li, Dan et al. (2014) Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit. Nat Struct Mol Biol 21:1028-34
Jishage, Miki; Malik, Sohail; Wagner, Ulrich et al. (2012) Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II. Mol Cell 45:51-63
Chen, Wei; Roeder, Robert G (2011) Mediator-dependent nuclear receptor function. Semin Cell Dev Biol 22:749-58
Malik, Sohail; Roeder, Robert G (2010) The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation. Nat Rev Genet 11:761-72