Date of Award

Spring 1989

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

ADR1, a transcriptional activator from the yeast Saccharomyces cerevisiae, is required for activation of the glucose-repressible alcohol dehydrogenase, ADH II (encoded by the ADH2 gene). The ADR1 gene encodes a protein which binds to an upstream activation sequence in the ADH2 promoter. Several methods were used to locate functional regions of the ADR1 protein.

The adr1-1 mutation was identified as a C to G transversion resulting in a nonsense codon at the eleventh codon of ADR1. tRNA-suppressors which substituted an amino acid at the eleventh codon of adr1-1 resulted in a functional adr1-1 protein, indicating that the translational start of ADR1 occurs at the first AUG of the ADR1 transcript. The adr1-1 5.1 kb RNA was found to be two- to three-fold less abundant than the ADR1 transcript. This observation is a further instance of a nonsense mutation early in a gene leading to decreased stability of the RNA and confirms that the 5.1 kb transcript is the ADR1 mRNA.

A series of C-terminal truncations of the ADR1 gene were constructed and used to investigate functional regions of ADR1. A C-terminal truncation leaving only 151 N-terminal amino acids (ADR1-151) and depleting part of a "Zn-binding" DNA-binding finger motif was not capable of transcriptional activation. In contrast, the ADR1-220 protein containing the complete "Zn-binding" DNA-binding finger motif was capable of transcriptional activation. However, none of the truncated ADR1 proteins were as transcriptionally active as the entire ADR1 protein.

An ADR1 protein containing at least 506 N-terminal amino acids was required for glycerol growth. Mutations in the GLP1 gene were identified that allowed strong glycerol growth, while remaining dependent on the presence of a functional ADR1 gene. This newly identified function of ADR1 suggests that ADR1 has a more extensive role in the regulation of nonfermentative growth in S. cerevisiae than previously expected.

Download

Share

COinS