The Leu3 protein: A metabolite-dependent transcriptional activator that is also a repressor
Abstract
The Leu3 protein (Leu3p) of Saccharomyces cerevisae is a transcriptional regulator. Its target is a 12-base-pair cis-acting element (UAS$\sb{\rm LEU})$ found upstream of several genes involved in branched-chain amino acid biosynthesis and nitrogen metabolism. Yeast strain XK160 was constructed for overproducing Leu3p from the GAL1 promoter. Sustained overproduction of Leu3p required elevated intracellular levels of $\alpha$-isopropylmalate ($\alpha$-IPM), the first intermediate of leucine biosynthetic pathway. Leu3p was purified 245,000-fold over wild type level by a procedure that included polyethylenimine precipitation, ammonium sulfate fractionation, heat treatment and UAS$\sb{\rm LEU}$-affinity chromatography. Highly purified as well as crude preparations were found to form two specific Leu3p-UAS$\sb{\rm LEU}$ complexes, with the apparent dissociation constants of 1.1 nM and 2.6 nM, respectively. Purified Leu3p is a mixture of phosphorylated and non-phosphorylated forms. The native Leu3p exists as a dimeric molecule, irrespective of the state of phosphorylation. In vitro, purified Leu3p can replace endogenous Leu3p to activate transcription from a template containing UAS$\sb{\rm LEU}$ element(s) upstream of a CYC1 TATA box. The Leu3p dependent transcriptional activation was observed only when $\alpha$-IPM was present. When $\alpha$-IPM was omitted, the presence of Leu3p resulted in a 3-5 fold decrease of the transcription below the basal level. $\alpha$-IPM was also required for Leu3p to compete effectively with another transcriptional activator, GAL4-VP16, for limiting transcription factors. Based on in vitro studies, the transcriptional activation and repression functions are located in separable segments of Leu3p. A mutant that lacks the activation domain still acts as a repressor, as does a severely truncated Leu3p that contains little more than the DNA binding region. A tryptophan to alanine substitution within the activation domain does not diminish the activation strength. However, the substitution results in a partial $\alpha$-IPM independent activator, i.e. a weak but measurable activation can be obtained with the mutant protein in the absence of $\alpha$-IPM, indicating that an intact repression region can not inhibit the transcriptional activation function of Leu3p.
Degree
Ph.D.
Advisors
Kohlhaw, Purdue University.
Subject Area
Biochemistry|Molecular biology
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