Date of Award

Fall 1999

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde L Denis

Abstract

The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively. This study demonstrates that the CCR4-NOT complex functionally and physically interacts with TBP and TAFs. Firstly, mutations in CCR4, NOT4, and NOT5 suppressed the his4-912 delta insertion by a mechanism similar to that observed for the defective TBP allele spt15-122. This mechanism appeared to involve stabilization of TBP binding to a specific non-consensus TATA sequence, CATAAA, in the his4-912 delta element. Secondly, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, it was found that the NOT proteins were general repressors that disrupt TBP function irrespective of the DNA sequence. Thirdly, increasing the dosage of NOT 1 specifically inhibited the ability of spt15-122 to suppress the his4-912 delta insertion but did not affect the Spt-phenotype of either spt3 or spt10 at this locus. Fourthly, spt3, spt8 and spt15-21 alleles (all involved in affecting interaction of SPT3 with TBP) suppressed ccr4 and caf1 defects. Also, a number of genetic interactions were observed between CCR4-NOT complex components and TBP and TAFs.

In addition, NOT2 and NOT5 were found to physically interact with TBP, multiple TAFs, and SPT3. Moreover, NOT2 and NOT5 were found to associate with components from purified SAGA and TFIID complexes, respectively. These genetic an physical interactions indicate that one role of the CCR4-NOT complex is to inhibit functional TBP-DNA interactions, perhaps by interacting with and modulating the function of factors which associate with TBP such as the components of TFIID and SAGA.

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