| Budget Amount *help |
¥3,820,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
In Saccharomyces cerevisiae the expression of genes involved in the synthesis of thiamin pyrophosphate and the utilization of thiamin from the extracellular environment (THI genes) is coordinately induced in response to thiamin starvation, a mechanism called the yeast THI regulatory system. In this system thiamin pyrophosphate (TPP) serves as a corepressor, and three positive regulatory factors (Thi2p, Thi3p and Pdc2p) have been identified. Thi3p senses the intracellular signal, TPP, and there exists a putative DNA-binding domain in Thi2p and Pdc2p. However, it is not known how these regulatory factors function in the signal transduction pathway leading to the transcriptional regulation of THI genes.
In the previous year, we found that Pdc2p has the transcriptional activity and interacts with Thi3p, and that these two activities are partially diminished by exogenous thiamin. In addition, the transcriptional activity of Pdc2p is decreased in thi3 disruptant. Then we tried to determine the functional domains of Pdc2p (926 amino acids) using a series of deletion mutants. It was suggested, by yeast one-hybrid and two-hybrid analyses, that the regions from 407 to 581 and that from 582 to 889 are necessary for the transcriptional activity and the interaction with Thi3p, respectively. When the Thi3p-interacting domain was removed from Pdc2p, the transcriptional activity was unaffected by the defect of THI3 gene. Moreover, a fluorescence quenching study with acrylamide was carried out on a sole tryptophan residue (Trp-450) of recombinant proteins, Pdc2p(407-581) and Pdc2p(407-926). The removal of the 582-926 region from Pdc2p(407-926) resulted in an increase in accessibility of the quencher, suggesting that the Thi3p-interacting domain masks the transcriptional activation domain. It is likely that the interaction with Thi3p causes a conformational change in the Pdc2p-DNA complex leading to exposure of the transcriptional activation domain to solvent.
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