An investigation of the transcriptional regulation of thiamin biosynthesis in Saccharomyces cerevisiae
Baker's yeast Saccharomyces cerevisiae can synthesise vitamin B1, thiamin, de nova. Exogenously supplied vitamin is, however, taken up very efficiently, leading to high intracellular levels of the enzyme cofactor thiamin diphosphate (ThDP) and subsequent repression of genes encoding the biosynthetic enzymes. This project examines the mechanisms by which the small thiamin molecule is able to regulate transcription.;To date, three transcriptional activators (Thi2p, Pdc2p and Thi3p) of thiamin biosynthesis have been identified. To allow the sub-cellular localisation of these proteins, they were tagged at the C-termini with GFP. Fluorescence microscopy of cells expressing these fusion proteins has revealed that Thi2p, Thi3p and Pdc2p all localise to the nucleus under both repressing (growth in medium containing thiamin) and derepressing (growth in medium lacking thiamin) conditions.;The TH14 gene, which is negatively regulated by ThDP and encodes an enzyme involved in the biosynthesis of the co-factor, is used as a model for all thiamin-regulated genes. A detailed analysis of the TH14 promoter was carried out in order to identify upstream activator and/or repressor sequences. A series of TH14-promoter truncations. deletions and translocations were used to drive expression of a LacZ reporter gene. This led to the identification of a bp region of the TH14 that contains an activator sequence necessary for high-level expression.;Electrophoretic mobility shift assays were performed on various TH14-promoter fragments using crude yeast cell extracts. A protein complex could be detected binding to a region of the promoter close to the activator sequence. This complex forms in a sequence specific manner and is present when using extracts from cells grown in both the presence and absence of thiamin. Moreover, deletion of the activator sequence did not affect complex formation. The complex also appears not to contain any of the known regulators of thiamin biosynthesis. It remains unknown whether this complex plays any direct role in the regulation of TH14 transcription.