|Keywords:||transcription; non-coding RNA; Mot1p-NC2; TATA-binding protein; chromatin; SAGA; TFIID|
|Full text PDF:||http://dspace.library.uu.nl:8080/handle/1874/308652|
Transcription underlies all cellular processes and responses to internal and external cues. Deregulation of transcription has implications for the fitness of the cell or organism. During my PhD I have investigated the importance of proper TATA-binding protein (TBP) regulation as a mechanism to ensure transcription fidelity in the model organism Saccharomyces cerevisiae. Gene transcription in eukaryotes is performed by three nuclear multi-subunit DNA-dependent RNA polymerases, pol I, pol II and pol III, which synthesize RNA from a DNA template. The three RNA polymerases transcribe distinct sets of genes. Pol II produces messenger RNAs (mRNAs) and deserves the largest diversity of gene promoters. The transcription cycle starts with assembly of the pre-initiation complex (PIC) on promoters of genes. The PIC consists of pol II and six basal transcription factors. PIC nucleation starts with binding of TBP to TATA-like sequence in the core promoter. Transcription initiation by pol II is restricted to nucleosome-depleted regions (NDRs) of the yeast genome, which are accessible for PIC components like TBP and TFIID. Transcriptional output of coding genes is determined by balancing TBP recruitment by the Spt-Ada-Gcn5-acetyltransferase (SAGA) or TFIID complexes with TBP displacement by the Swi2/Snf2-like ATPase Mot1p and the negative cofactor 2 (NC2) complex consisting of NC2a and NC2b I studied genome-wide regulation of gene expression by Mot1p and NC2. I observed differential expression responses on the two gene classes of pol II, by Mot1p and NC2 action. Mot1p establishes a dynamic pool of TBP by removing TBP from intrinsically preferred TATA-containing binding sites by the use of energy derived from ATP hydrolysis and redistributes TBP to low affinity TATA-less (or TATA-like) sites. TFIID-dependent TATA-less genes are activated and SAGA-dependent TATA-containing genes are repressed. This explains how Mot1p and NC2 repress SAGA-dependent TATA-containing genes and activate TFIID-dependent TATA-less genes. I discovered a novel function for Mot1p and NC2 and showed that these factors play “hidden” roles in ensuring transcription fidelity by restricting ncRNA synthesis.They operate to counteract TBP binding and PIC formation at spurious sites and to prevent intragenic and anti-sense ncRNA expression. The negative TBP regulators cooperate of with specific chromatin regulators to suppress intragenic transcription and they restrict antisense ncRNA production by inhibiting TBP binding and PIC formation in gene bodies and at the 3’-end of genes. Together, these results provide novel mechanistic insight in the regulatory mechanisms that limit the accumulation of non-coding RNAs in the yeast genome. Overall, the picture appears that Mot1p and NC2 have a global role in surveilling the genome. My research adds nvel knowledge of mechanisms that ensure transcription fidelity.