The long term goal of this proposal is to define the mechanisms by which protein-protein interactions control gene expression. Noncovalent bridges between DNA-binding factors and RNA polymerases are made by a recently-discovered class of proteins called coactivators. Key questions about coactivators include: How do they recognize specific transcription factors? What steps in transcription do they regulate? How are coactivators regulated? To address these issues at the molecular level, this proposal focuses on x-ray structural studies of DCoH, the dimerization cofactor of hepatocyte nuclear factor 1 (HNF-1), and its complex with HNF-1alpha. DCoH binds tightly to the dimerization domain of the HNF-1 family of DNA-binding, liver-specific, transcription factors and enhances by over 200-fold expression from HNF-1-dependent genes. DCoH also functions in contexts that lack HNF-1, suggesting more catholic protein interactions. In addition, DCoH catalyzes the dehydration of an essential cofactor of aromatic amino acid hydroxylases. To investigate the connections between the transcriptional and enzymatic activities of DCoH and to define its interactions with HNF-1, Dr. Alber proposes the following specific aims: 1. Refine the high resolution x-ray crystal structure of rat DCoH; 2. Determine the structures of DCoH complexed with inhibitors of the enzyme activity; 3. Determine the high resolution structure of the dimerization motif of HNF-1alpha; and 4. Determine the structure of the complex of the HNF-1alpha dimerization domain and DCoH. These studies will have broad implications for the mechanisms of coactivator function. Because gene regulation plays a central role in many diseases-- including birth defects, viral infections and cancer--the proposed work has direct significance for molecular biology and medicine.
|Rose, Robert B; Pullen, Kristi E; Bayle, J Henri et al. (2004) Biochemical and structural basis for partially redundant enzymatic and transcriptional functions of DCoH and DCoH2. Biochemistry 43:7345-55|
|Crowder, S; Holton, J; Alber, T (2001) Covariance analysis of RNA recognition motifs identifies functionally linked amino acids. J Mol Biol 310:793-800|
|Endrizzi, J A; Beernink, P T; Alber, T et al. (2000) Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: implications for allosteric regulation. Proc Natl Acad Sci U S A 97:5077-82|
|Rose, R B; Endrizzi, J A; Cronk, J D et al. (2000) High-resolution structure of the HNF-1alpha dimerization domain. Biochemistry 39:15062-70|
|Beernink, P T; Endrizzi, J A; Alber, T et al. (1999) Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit. Proc Natl Acad Sci U S A 96:5388-93|
|Crowder, S M; Kanaar, R; Rio, D C et al. (1999) Absence of interdomain contacts in the crystal structure of the RNA recognition motifs of Sex-lethal. Proc Natl Acad Sci U S A 96:4892-7|