Biochemical and functional analysis of TBP-containing complexes

Abstract

The regulation of transcription initiation is a key feature of gene expression control. RNA polymerase II is recruited to the DNA upon assembly of the Pre-Initiation Complex (PIC), which is in turn nucleated by the binding of TATA-binding protein (TBP) to the core promoter. The modulation of TBP activity occurs in a dynamic fashion by controlling the TBP delivering and removal rates. TFIID and SAGA have a positive role on TBP activity whereas MOT1p and NC2 have a negative role. The interplay between the TBP-binding complexes TFIID, SAGA, MOT1 and NC2 regulates transcriptional output. In this thesis, a biochemical and functional analysis of TBP-binding complexes has been performed to decipher their roles in transcription initiation. Posttranslational modifications of the TBP-binding complexes TFIID and SAGA were mapped by mass spectrometry. This revealed that many residues on shared or specific subunits of the two complexes were subject to modification by phosphorylation or acetylation. The shared subunit Taf5p was phosphorylated when present in SAGA, but not when present in TFIID. The SAGA-specific subunits Spt7p and Sgf73p were hyperacetylated in chromatin binding domains. The SAGA complex is also subject to proteolytic cleavage of the Spt7p subunit resulting in formation of the related SLIK protein complex. Using mass spectrometry, we identified this cleavage site. A knock out screen combined with an in vitro protease cleavage assay resulted in the identification of the Pep4p protease to be responsible for cleavage of Spt7p and formation of SLIK. The proteolytic processing of the Spt7p subunit affects the composition of the TBP-module of SAGA by removing the binding domain for the Spt8p subunit. We have reviewed the current literature on SAGA in yeast, flies and human, and addressed how yeast SAGA has diverged into two related but distinct protein complexes in metazoans: SAGA and ATAC. Next, the genome-wide regulation of gene expression by Mot1p and NC2 has been studied using a new approach for conditional protein depletion in living yeast cells. This indicated that Mot1p and NC2 coregulate gene expression to a large extent. Both Mot1p and NC2 were required for maintaining basal gene expression and for the shut down of gene expression following induction by heat shock. Taken together, the results presented in this thesis have uncovered how the SAGA complex is converted to the SLIK complex; have identified a large number of post-translational modifications on SAGA and TFIID; and have provided further insight in the mechanism of gene repression by Mot1 and NC2. These findings should aid in our understanding of TBP function in the regulation of gene expression.