The broad objective of the proposed research is to obtain a molecular-level understanding of the interactions between transcriptional activators and their target proteins within the transcriptional machinery using a suite of chemical approaches. Misregulation of transcription is either a cause or an effect of human diseases such as cancer, metabolic disorders, and infectious diseases. Thus, molecules that inhibit or promote the protein-protein interactions that regulate transcription are highly desirable both as mechanistic probes and, in the long term, as therapeutic agents that correct transcriptional errors. Only a handful of such molecules have been reported, however, due in part to the still-limited understanding of the mechanism of transcriptional regulation. In the case of transcriptional activators, proteins that activate the transcription of particular genes in a signal- responsive fashion, little conclusive is known about either the interaction partners within the transcriptional machinery or the functionally relevant thermodynamic parameters. As part of Specific Aim 1, we will kinetically characterize the interactions between several transcriptional activators and their target proteins in order to parse the connection between levels of gene up-regulation and the rates of association and dissociation of activator-transcriptional machinery protein complexes.
In Specific Aims 2 and 3 in vivo (S. cerevisiae) crosslinking experiments along with FT-ICR mass spectrometric analysis of crosslinking products will be employed to identify the physiologically relevant targets of transcriptional activators, thus addressing a long-standing and fundamental mechanistic question in eukaryotic transcription. Taken together, the data generated through these experiments will provide a high resolution map of what and where transcriptional activators bind in the transcriptional machinery as they initiate gene up-regulation. This information will be enormously enabling for the discovery of transcription-targeting small molecules and, longer term, for the development of small molecules that target individual transcriptional activator-transcriptional machinery protein interactions implicated in human disease.

Public Health Relevance

The experiments outlined in this proposal will provide a detailed picture of the binding profile of transcriptional activators and the contribution that the individual binding events make to transcriptional regulation. These data will make it possible to design more effective screens of small molecules that function as activators or inhibitors of transcription and, further, will provide a strategy by which specificity can be quickly evaluated. Ultimately, this will facilitate the discovery and development of transcription-based therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065330-09
Application #
8065354
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Sledjeski, Darren D
Project Start
2002-04-01
Project End
2013-04-30
Budget Start
2011-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2011
Total Cost
$259,720
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Pomerantz, William C; Wang, Ningkun; Lipinski, Ashley K et al. (2012) Profiling the dynamic interfaces of fluorinated transcription complexes for ligand discovery and characterization. ACS Chem Biol 7:1345-50
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Krishnamurthy, Malathy; Dugan, Amanda; Nwokoye, Adaora et al. (2011) Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo. ACS Chem Biol 6:1321-6

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