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1.
EMBO J ; 19(12): 3016-27, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10856245

ABSTRACT

The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.


Subject(s)
Fungal Proteins/chemistry , Nuclear Proteins , Repressor Proteins/chemistry , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Fungal Proteins/genetics , Fungal Proteins/metabolism , Homeodomain Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Peptide Fragments/chemistry , Protein Binding , Protein Structure, Secondary , Repetitive Sequences, Amino Acid , Repressor Proteins/genetics , Repressor Proteins/metabolism , Saccharomyces cerevisiae
2.
J Biol Chem ; 275(12): 9011-8, 2000 Mar 24.
Article in English | MEDLINE | ID: mdl-10722750

ABSTRACT

The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Saccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repression in vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and (1)H NMR show that this domain of Tup1 is primarily alpha-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein.


Subject(s)
DNA-Binding Proteins , Fungal Proteins/metabolism , Nuclear Proteins , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Binding Sites , Candida albicans , Circular Dichroism , Crystallography, X-Ray , Fungal Proteins/chemistry , Fungal Proteins/genetics , Models, Molecular , Mutation , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Saccharomyces cerevisiae , Species Specificity , Surface Plasmon Resonance , Ultracentrifugation
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