A molecular switch: Structure-function study of a yeast regulatory protein LEU3

Kemin Zhou, Purdue University

Abstract

LEU3 is a regulatory gene responsible for the regulation of branched chain amino acid biosynthetic genes in yeast Saccharomyces cerevisiae. To understand how the LEU3 protein activates its target genes in response to $\alpha$-isopropylmalate ($\alpha$-IPM), I sequenced a 3.6 kilobase DNA fragment containing the LEU3 gene which encodes a protein of 886 amino acids and has two consensus GCN4-binding sites in the 5$\sp\prime$-noncoding region. To reveal the structural elements responsible for the three major functions: DNA-binding, transcriptional activation, and modulation defined as the ability of the LEU3 protein to become transcriptionally active in response to $\alpha$-IPM, I created and characterized deletion, insertion, and point mutations of LEU3 using a LEU2/lacZ fusion as a reporter for the activation and modulation functions of the LEU3 protein. The DNA-binding activity of LEU3 mutants was monitored with gel mobility-shift assays. Two functional domains are located on the termini of the LEU3 protein. The DNA-binding activity was located in the N-terminal 99 amino acids. This region contains a cysteine-rich motif (C6 motif) that may form a zinc-cluster structure. The activation function was located in the C-terminal 32 amino acids which are rich in negatively charged residues. The modulation domain was located in the central part (174-773) of the LEU3 protein. Amino acids between nay domains are dispensable for the known functions of LEU3. The mechanism of modulation is postulated to occur through dissociation and association of the activation and the modulation domains in response to the presence and absence of $\alpha$-IPM. Of six acidic residues in the activation domain, two aspartate and a glutamate are especially important for the activation activity of the LEU3 protein. When regarded as an allosteric transcriptional regulator, LEU3 can be described by a set of parameters describing allosteric proteins. In these terms, most point mutations in the activation domain reduced the intrinsic activation function. The modulation function is even more sensitive to point mutations.

Degree

Ph.D.

Advisors

Kohlhaw, Purdue University.

Subject Area

Molecular biology|Biochemistry

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