Transcriptional regulation of yeast LEU4 and GDH1 genes and the LEU3 protein

Yuanming Hu, Purdue University

Abstract

The LEU4 gene of Saccharomyces cerevisiae encodes $\alpha$-isopropylmalate synthase, which catalyzes the committed step in leucine biosynthesis. The reaction product $\alpha$-isopropylmalate also functions as co-activator for the transcriptional factor Leu3p. The metabolic importance of $\alpha$-isopropylmalate appears to be reflected in the multifaceted regulation of LEU4 expression. Deletion and mutational analysis of the LEU4 promoter revealed five functional cis elements. Three distal elements, UAS$\rm\sb{LEU},$ GCE-A and GCE-B, are responsible for regulation by Leu3p and Gcn4p, respectively. Their actions are independent and approximately additive. Two proximal elements were also localized. One of these exhibits high affinity for TATA binding protein and is essential for LEU4 expression. The other element shows strong sequence identity with the Bas2p binding site and appears to be involved in the basal regulation of LEU4. The promoter of GDH1, the gene encoding NADP$\sp+$-dependent glutamate dehydrogenase (NADP-GDH), contains a UAS$\rm\sb{LEU}$-homologous sequence for Leu3p. First I demonstrate that Leu3p specifically interacts with this UAS$\rm\sb{LEU}$-homologous sequence. Then I show that Leu3p is required for full activation of GDH1. Mutation of LEU3 and deletion of UAS$\rm\sb{LEU}$ both reduced the expression of GDH1 significantly. The involvement of Leu3p in GDH1 expression is also reflected in the GDH1 mRNA level and NADP-GDH activity. I also demonstrate that GDH1 expression correlates with the cells' ability to generate $\alpha$-isopropylmalate. I conclude that Leu3p is a more general transcription regulator. Leu3p is a pleiotropic transcription regulator. Leu3p is unique in that transcription activation by Leu3p requires an intermediate $\alpha$-IPM. To tackle the $\alpha$-IPM-mediated modulation and activation functions of Leu3p I tried to isolate activation-deficient mutants for genetic analysis. I first cloned and identified mutations of three spontaneous leu3 mutants. All three leu3 mutants had single nucleotide mutations resulting in activation domain deletions. Then, I created a mutant in which two aspartates in the activation domain were changed to asparagines. This mutant Leu3p behaved as a constitutive repressor and is a modulation-deficient mutant. The importance of this double mutant is discussed.

Degree

Ph.D.

Advisors

Kohlhaw, Purdue University.

Subject Area

Molecular biology

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