One long term objective of this application is to detail the molecular mechanism of transcriptional repression of the hypoxic genes in Baker's yeast, a model eukaryote. Repression of these genes involves the binding of a specific repressor protein Rox1 to operator sequences. Rox1 recruits the Ssn6/Tup1 general repression complex to the locus. This yeast system may provide insights into the cellular response to hypoxia in humans, an important aspect of tissue recovery from wounds and surgery. On a more general level, transcriptional repression plays an important role in regulating gene expression in all organisms. Recently, mutations in genes with homology to Tup1 have been implicated in the inherited human diseases Cockayne's, DiGeorge, and Shprintzen Syndromes, and familial congenital heart disease suggesting a conservation of regulatory mechanisms in eukaryotic systems. A second long term objective is to explore the structure/function relationship in the HMG-architectural protein Rox1. HMG DNA binding and bending proteins are highly conserved in sequence and their DNA binding sites. This class includes the SRY and SOX sex-determining protein of humans, mutations in which cause varying degrees of XY sex reversal. Knowledge of how these proteins bind and bend DNA would help in designing proteins that would introduce predictable topology into DNA. The experimental approaches combine genetic and biochemical analyses. Mutational analysis of the ROX1 gene will identify proteins with altered DNA binding and bending specificity. These mutant proteins will be characterized and their NMR structure determined. Also, the mutational analysis of ROX1, along with a mutational analyses of the SSN6 and TUP1 genes, will detail the interactions among their products. Biochemical assays for these interactions will be developed. Suppressor analyses will define other cell components, presumably of the transcriptional machinery, that interact with complex. Also, studies are proposed to identify the function of eIF5A, encoded by aerobic/hypoxic homologues, a putative translational initiation factor recently implicated in protein transport, and of Rox3, an essential, nuclear protein, in the global stress response.
Showing the most recent 10 out of 15 publications
