Date of Award

Spring 2000

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde L Denis

Abstract

The mechanisms of transcriptional regulation are well conserved from yeast to human cells. Most genes are regulated at the transcriptional level, particularly, at the transcription initiation step. In Saccharomyces cerevisiae at least one transcriptional activator, ADR1, and one transcription complex, CCR4-NOT, participate in the activation of the ADH2 gene. The core 1 x 106 dalton CCR4-NOT complex consists of CCR4, CAF1, the five NOT proteins (NOT1-NOT5), and three other uncharacterized proteins. Deletion of NOT3, NOT4, or NOT5 did not affect the association of CCR4 with CAF1 in the gel filtration analysis. In contrast, NOT2 inactivation caused partial destabilization of the CCR4-NOT complex. In addition, deletion or mutation in the NOT1 N-terminal resulted in the dissociation of CCR4 and CAF1 from the complex. TFIID functions in the first step in initiating transcription by binding the TATA-box. To investigate interactions of ADR1 with TFIID in detail, a series of binding assays between ADR1 transcriptional activation domains (TADs) and TFIID components were conducted to identify the individual TAF proteins that interact with ADR1 directly. Multiple TAF proteins were found to bind three of the four TADs of ADR1. TAF150/TSM-1, TAF130/145, TAF60 and TAF61/68 proteins were found to bind TADIV. The region of TAF150 that bound TADIV was located to a nonconserved region (residues 744--1100) while the sequence of TAF130 that alone is capable of binding TADIV was localized to an essential region (residues 668--865) that is well conserved among TAF130 homologs. The physiological roles of these TAF-ADR1 interactions were further investigated using S1 nuclease analysis. An intact TFIID complex was shown to be required for ADH2 gene expression. Individual mutations in TAF150, TAF61, and TAF60 were also shown to reduce ADH2 transcription. Therefore, just as other activators display multiple physical interactions with core transcriptional components, ADR1 also displayed multiple apparently redundant interactions. These biochemical interactions may benefit cells through redundancy and/or may function in a sequential or conditional way in response to distinct signal transduction pathways.

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