A proteomic screen to identify novel SUMO (small ubiquitin-related modifier) substrates in budding yeast Saccharomyces cerevisiae revealed the TATA-binding protein (TBP) as a candidate. TBP is a ubiquitous transcription factor required for transcription by the three eukaryotic RNA polymerases. TBP was confirmed to be SUMOylated in vivo using affinity purification techniques. Lysine (K) residues in the N terminus were confirmed to be the SUMO-accepting sites, suggesting the existence of a 'SUMO region' in the N-terminal domain of TBP. Among the six lysine residues identified, K47 was a strong SUMOylation site. I generated TBP SUMO-deficient mutants to interrogate the functional role of TBP SUMOylation. The mutants showed much higher protein stability. Additionally, chromatin immunoprecipitation (ChIP) analysis revealed that the occupancy of TBP SUMO mutants at promoters of both constitutive and inducible genes were much lower than TBP wildtype, with a lower recruitment efficiency. Cross-linking kinetic (CLK) analysis further proved that the lower promoter occupancy of the SUMO mutants was caused by lower promoter dynamics. This was supported by fluorescence recovery after photobleaching (FRAP) assays, which showed a larger immobile population for the TBP SUMO-deficient mutants. The lower promoter dynamics is expected to lead to lower transcription plasticity and/or aberrant transcriptional output. Consistent with this, cells expressing either type of TBP mutant exhibit significant defects during sporulation, which is a cellular process involving genome-wide transcription reprogramming.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:656546 |
| Date | January 2014 |
| Creators | Liu, I-Chun |
| Contributors | Aragon, Luis |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/24440 |
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