MECHANISMS OF THE SAGA COACTIVATOR COMPLEX IN REGULATION OF GENE TRANSCRIPTION
Open Access
- Author:
- Sermwittayawong, Decha
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 04, 2006
- Committee Members:
- Song Tan, Committee Chair/Co-Chair
Jerry L Workman, Committee Member
Benjamin Franklin Pugh, Committee Member
Joseph C. Reese, Committee Member
Robert Paulson, Committee Member
Juliette T J Lecomte, Committee Member - Keywords:
- Gcn5
competition
Spt3
TBP
Spt8
SAGA
bromodomain - Abstract:
- The SAGA coactivator complex functions in response to an activator protein to activate gene transcription. This thesis describes my investigations into two major functions of the SAGA complex: 1) its recruitment of the TATA-binding protein (TBP) and 2) its histone acetyl transferase activity (HAT) on a nucleosome substrate. My experiments show that the SAGA complex utilizes the Spt8 and possibly Ada1 subunits to bind the TATA-binding protein (TBP). This is the first time that Ada1 has been identified as a potential SAGA subunit that interacts directly with TBP. In contrast, Spt3, a strong genetic candidate for interacting with TBP, does not bind TBP on its own. However, in the context of the SAGA complex, Spt3 contributes to the overall interaction between TBP and SAGA. Sequence analysis indicates a putative WD40 domain repeats within the C-terminal region of Spt8. These putative WD40 repeats are apparently sufficient for the interaction with TBP. Unexpectedly, I find that Spt8 or the SAGA complex binding to TBP competes with TBP dimer formation. Furthermore, the association of Spt8 or the SAGA complex with TBP prevents TBP binding to the TATA box DNA, suggesting that Spt8 binds to the concave surface of TBP. Based on these results, I propose a hand off model that can explain why the SAGA complex is able to act alternatively as a corepressor or a coactivator. The hand off model also explains why the SAGA complex is not found localized to the core promoter in vivo. I have also investigated the requirement of the Gcn5 bromodomain for nucleosomal acetylation function by the trimeric Ada3/Ada2/Gcn5 SAGA subcomplex or the intact SAGA complex. My mutational analyses show three basic residues, K415, R419, and K422 that lie on the surface of the bromodomain, are necessary for the nucleosomal acetylation of the Ada3/Ada2/Gcn5 complex. Interestingly, the spatially distinct peptide binding pocket of the bromodomain is not necessary for the HAT function of the trimeric subcomplex. The Gcn5 bromodomain is also required for the global acetylation of histone H3 in yeast cells. Spt8, Spt3, and possibly Ada1 SAGA subunits function to regulate interaction with TBP, whereas the Gcn5 bromodomain is required for nucleosomal acetylation of the trimeric Ada3/Ada2/Gcn5 or the SAGA complexes. My results advance our basic understanding of how SAGA regulates gene transcription.