## Doctoral Dissertations

Spring 1993

Dissertation

Biochemistry

#### Degree Name

Doctor of Philosophy

Clyde L Denis

#### Abstract

Glucose repression of the ADH2 gene from Saccharomyces cerevisiae is mediated by the abundance and activity of the transcriptional activator ADR1. The focus of this dissertation is to characterize the mechanisms by which glucose controls the ability of ADR1 to activate ADH2 transcription.

Glucose results in a two-fold decrease in the steady state levels of ADR1 mRNA. This glucose-dependent reduction in steady state ADR1 mRNA was shown to be due to an increased rate of ADR1 mRNA degradation. The unusually long, 510 nucleotide 5$\sp\prime$ untranslated region of the ADR1 mRNA appeared to mediate this glucose-dependent ADR1 mRNA decay.

To better understand the posttranslational control of ADR1 activity by glucose, the ADR1$\sp{c}$ mutations which allow glucose-insensitive ADH2 expression were analyzed. The mechanism by which three mutated genes (saf1, saf2, and saf3) suppressed the ADR1$\sp{c}$ phenotype was investigated. Each of the mutated saf genes was found to suppress ADR1$\sp{\rm c}$ activity by reducing ADR1$\sp{c}$ transcription 5- to 8-fold under glucose growth conditions. The SAF genes were also required for ADR1 transcription under glucose conditions, indicating that these genes are not specifically involved in ADR1$\sp{\rm c}$ function. A deletion analysis conducted on an ADR1$\sp{\rm c}$ protein indicated that no ADR1 residues outside the site of the ADR1$\sp{\rm c}$ mutations were specifically required for ADR1$\sp{\rm c}$ function. ADR1$\sp{c}$ mutations enhanced the activity of ADR1 proteins that contained either of two separate activation domains.

Deletion analysis also allowed for the improved mapping of the functional domains in ADR1. ADR1 was found to contain multiple domains required for transcriptional activation, and these activation domains were found to be separated by residues that inhibit activation. One inhibitory region encompasses the site of the ADR1$\sp{\rm c}$ mutations. This array of alternating activating and inhibitory regions in ADR1 suggests that ADR1 activity is modulated by interactions between different positive and negative domains in ADR1.

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