Self-silencing of yeast transcriptional activator Leu3p: Studies on mechanism, specificity and regulation of the activation domains masking process
Abstract
The Leu3 protein of Saccharomyces cerevisiae regulates the transcription of genes involved in branched-chain amino acid biosynthesis and in ammonia assimilation. Leu3p can either activate or repress gene transcription depending on the intracellular level of α-isopropylmalate, an intermediate of the leucine biosynthesis pathway. This functional control process is called modulation. Leu3p modulates its function through masking/unmasking its activation domain. The observation that Leu3p retains its modulation properties even when expressed in mammalian cells suggests that this process requires no participation of auxiliary factors. This thesis addresses the molecular mechanism of the activation domain masking process. Characterization of an activation domain mutant (D872N/D874N), designated Leu3-dd, shows that it is an inactive molecule because its activation domain is permanently masked. A modified yeast two hybrid experiment demonstrates that the activation domain indeed interacts with another portion of Leu3p. Experiments of domain swapping between Leu3p and Cha4p (another yeast transcription factor involved in serine/threonine degradation) further delimits the activation domain masking region to the middle region of Leu3p, and indicates that the masking interactions between the activation domain and the middle region are highly specific. To identify the key residues involved in activation domain masking, the phenotype of Leu3-dd containing cells (very slow growth on SD plates) was used to select for suppressor mutations in the middle region. Eight amino acids are identified and allocated in a relatively small region. When anyone of them is mutated, the activation domain masking interactions between the activation domain and the middle region are essentially abolished, and the mutant Leu3p behaves as a “constitutively” active molecule. Finally, it is demonstrated that in vivo Leu3p can be modulated by its own concentration, suggesting a second mechanism to regulate the activation domain masking process.
Degree
Ph.D.
Advisors
Kohlhaw, Purdue University.
Subject Area
Molecular biology|Genetics|Microbiology
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