ABSTRACT
The Saccharomyces cerevisiae Tup1 protein is a member of a family of WD repeat containing proteins that are involved in repression of transcription. Tup1, along with the Ssn6 protein, represses a wide variety of genes in yeast including cell type-specific and glucose-repressed genes. Tup1 and Ssn6 are recruited to these specific gene sets by interaction with sequence-specific DNA binding proteins. In this work, a protein complex containing Ssn6 and Tup1 was purified to determine its composition. The size of the complex is estimated to be 440 kDa. Tup1 and Ssn6, which are both phosphoproteins, are the only proteins present in stoichiometric amounts in the complex. We also demonstrate that this purified complex represses transcription in an in vitro assay.
Subject(s)
DNA-Binding Proteins , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Nuclear Proteins , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Transcription, Genetic , Cell-Free System , Fungal Proteins/isolation & purification , Molecular Weight , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Protein Binding , Recombinant Fusion Proteins/metabolism , Repressor Proteins/isolation & purification , Saccharomyces cerevisiaeABSTRACT
The alpha 2 protein, a homeodomain protein involved in specifying cell type in the budding yeast Saccharomyces cerevisiae, is a transcriptional repressor. alpha 2 binds cooperatively with Mcm1, a serum response factor-related protein, to the a-specific gene operator. The alpha 2-Mcm1 complex in turn recruits Ssn6 and Tup1 to the operator, and we believe that these latter two proteins are responsible for the transcriptional repression. Placement of the a-specific gene operator in any of a variety of positions upstream of a test promoter leads to repression of that promoter in vivo. In this respect, the a-specific gene operator resembles a negatively acting enhancer. Here we describe the in vitro reconstitution of this example of negative control from a distance. We observe repression in vitro in the absence of exogenously added activator protein and on templates that lack binding sites for known activator proteins, and we infer that alpha 2-directed repression acts on the general transcription machinery.
Subject(s)
DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Homeodomain Proteins , Nuclear Proteins , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Transcription, Genetic , Escherichia coli , Operator Regions, Genetic , Point Mutation , Recombinant Proteins , Saccharomyces cerevisiae/genetics , Templates, GeneticABSTRACT
The authors analyse the technical problems encountered in using E.E.G. in children's reanimation. They show some examples which illustrate the importance of E.E.G. in diagnosis and therapy of such cases. Emphasis is laid upon the need for close collaboration between the EEGers and the reanimation staff.
