This project addresses the function of the yeast CYC8 (SSN6) and TUP1 proteins, which appear to play a novel role in transcriptional repression. The mechanism of repression of transcription in eucaryotic cells is poorly understood. Recent evidence has shown that products of two yeast genes, CYC8 (SSN6) and TUP1, are required for the function of several repressors including those controlling glucose repression (MIG1) and mating type (alpha2 and alpha1-alpha2). Mutations in either gene produce constitutive expression of many glucose-repressible genes, but the exact functional role of the CYC8 and TUP1 gene products is unknown. The TUP1 protein has six repeats with strong homology to the b-subunit of heterotrimeric G-proteins, and the CYC8 protein contains 10 repeats of the tetratricopeptide (TPR) type which have been found in proteins associated with the nuclear scaffold. Both of these repetitive domains have been postulated to mediate protein-protein interactions. We have demonstrated that the CYC8 and TUP1 proteins are associated in a large multi-subunit complex. In order to identify the functional domains of the TUP1 protein, systematic mutagenesis employing linker-scanning and in-frame internal deletions will be performed on the protein-coding region. The mutant TUP1 proteins will be expressed in yeast cells to test for function and in vitro to test for association with the CYC8 protein. We will specifically address the roles of the TPR and beta-transducin repeats in the formation of the CYC8-TUP1 complex. We will perform experiments to distinguish between two likely mechanisms for the dependence of MIG1 repression on CYC8-TUP1: 1) the CYC8-TUP1 complex represses transcription by contacting he MIG1 repressor bound to DNA; 2) the CYC8-TUP1 complex acts indirectly by facilitating activation or DNA-binding of the MIG1 repressor. An understanding of the functions of CYC8 and TUP1 should provide insight into how eucaryotic cells can coordinately regulate transcription of a large set of genes, which is a critical feature of cancer, multicellular development, and cellular responses to hormones.