Characterization of the N-terminal domain of the yeast transcriptional repressor Tup1 - Proposal for an association model of the repressor complex Tup1 center dot Ssn6

The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown t hat the N-terminal domain of the Tup1 protein interacts with a region of th e Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide mot if. In this work, we use a surface plasmon resonance assay to measure the a ffinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Sacc haromyces cerevisiae SsnG that is sufficient for binding to Tup1. This doma in of SsnG binds with comparable affinity to S. cerevisiae and Candida albi cans Tup1, but with 100-fold lower affinity to Tup1 protein containing a po int mutation that gives rise to a defect in repression in vivo. Results fro m studies using analytical ultracentrifugation, CD spectroscopy, limited pr oteolysis, and H-1 NMR show that this domain of Tup 1 is primarily alpha-he lical and forms a stable tetramer that is highly nonglobular in shape. X-ra y diffraction recorded from poorly ordered crystals of the Tup1 tetrameriza tion domain contains fiber diffraction typical of a coiled coil. Our result s are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the SsnG protein.