Date of Award

Winter 1993

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde Denis

Abstract

The regulation of the ADH2 gene from the yeast Saccharomyces cerevisiae is carbon source-dependent. Expression of ADH2 requires the activity of the transcriptional activator ADR1. This dissertation addresses three separate aspects of the regulation of ADR1 activity.

We have identified eleven additional ADR1$\sp{c}$ mutations which allow ADH2 to partially escape glucose repression. These eleven new mutations cluster to one region of the ADR1 peptide between amino acids 227 and 239, previously identified by four other ADR1$\sp{c}$ mutations. These are the only mutations in ADR1 which have been isolated by virtue of their ability to activate ADH2 transcription under repressed conditions. These mutations have previously been shown not to affect ADR1 mRNA or protein levels suggesting that they likely affect the ability of ADR1 to interact with other proteins. This may entail relief of repression or an increased ability to interact with other transcription factors.

We have used an in vivo transcription assay to identify three distinct regions of the ADR1 protein which are capable of transactivation function. The location of these activation domains coincide with regions of the protein that were shown to be important for activation function by previous deletion analyses. We also identified two regions which display repressor function with this assay. One of these regions corresponds to the site of the ADR1$\sp{c}$ mutations.

In a modified in vivo transcription assay, we show that the UAS2 element, located adjacent to the binding site for ADR1 in the ADH2 promoter, is able to repress the activity of LexA-ADR1 fusions containing various activation domains of ADR1 as well as diminishing the activity of other transcriptional activators. The extent of repression varied depending on the particular activation domain of ADR1 present. Under derepressed conditions, inclusion of the UAS2 element in a hybrid promoter containing the LexA binding site increased the activity of the promoter when coexpressed with LexA-ADR1 fusions. This increase in activity was not specific for ADR1 as we also observed an increase in activity when other activators were bound to the template. These results suggest that ADR1 transcriptional activity is the mediated by complex interactions between activation and inhibition domains located within the molecule and that the ability of ADR1 to function at the ADH2 locus is governed by other factors which bind to the promoter region at UAS2.

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