Date of Award

Winter 1997

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde Denis

Abstract

The purpose of these studies was to examine the mechanism of function of the transcriptional activator ADR1 of yeast Saccharomyces cerevisiae. ADR1 is required for the activation of the ADH2 gene, as well as genes involved in glycerol metabolism and peroxisome biogenesis. ADR1 is a DNA-binding protein. It binds to a 22 bp palindromic upstream activating sequence 1 (UAS1) element located 215 bp 5$\sp\prime$ to the transcription start site of ADH2. Elements similar to UAS 1 are found in promoters of all ADR1-dependent genes. ADR1 was shown previously to contain three transcriptional activation domains (TADs): TADI (amino acids 76-172), TADII (263-357) and TADIII (420-462).

A novel activation domain, TADIV (residues 642-704), was identified in ADR1. Analysis of different derivatives of TADIV indicated that its activation function is principally localised to residues 698-704. In contrast to deletion of other ADR1 activation domains, deletion of activation domain TV from ADR1 severely compromised its ability to activate ADH2 transcription.

ADR1 activation domains have been shown to directly interact in vitro with ADA2 and GCN5 components of the ADA2 co-activator complex, and both ADA2 and GCN5 are also required for full ADH2 derepression. In addition, direct interaction of ADR1 TADs with TFIIB was observed, in which TADI displayed the strongest binding. A point mutation in TFIIB that reduced ADH2 derepression was found to result in decreased in vitro interaction of TFIIB with TADI. These results suggest that TFIIC is a functional contact for ADR1 activation of transcription.

Defects in ADA2, GCN5 and TFIIB do not severely reduce ADR1 ability to activate transcription, suggesting that there may be additional contacts that ADR1 makes with the components of transcriptional machinery. In order to identify these proteins, I examined which known components of the general transcription machinery are retained by the ADR1 activation domains from yeast whole cell extracts. It was found that ADR1 transcription activation domain IV (TADIV) specifically retained core transcription factor IID. Moreover, ADR1 could be co-immunoprecipitated with the TAF90 component of the TFIID from yeast whole cell extracts. ADH2 activation by ADR1 required the presence of intact TFIID in vivo, suggesting that the physical interactions that were observed had functional relevance.

We propose that multiple contacts made by the ADR1 activation domains to different components of the general transcriptional machinery and to the ADA2 co-activator complex contribute synergistically to the activation of the ADH2 gene. After facilitating the first rate limiting step--for example, TFIID recruitment, other steps in the preinitiation complex assembly become rate limiting, and even though transcription rate may have already become significant, it is further increased by ADR1 establishing new contacts to TFIIB and possibly other general transcription factors.

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