No such enhancement was observed in the thi3Δ strain. However, Pdc2p expressed striking transactivation activity in a Thi3p-independent fashion when the C-terminal region containing the Thi3p-interacting domain was shortened (Nosaka et al., 2008). Based on these observations, we proposed a mechanism for the transcriptional activation of THI genes mediated by Pdc2p in response to thiamin starvation as follows. When intracellular learn more TPP is abundant and occupies the TPP-binding sites of Thi3p, the C-terminal domain of Pdc2p masks the internal domain responsible for the transactivation activity. Upon thiamin deprivation,
the dissociation of TPP from Thi3p is followed by the interaction of Thi3p with the C-terminal domain
of Pdc2p, which in turn causes a conformational change in Pdc2p. As a result, the C-terminal domain is removed from the transactivation domain; thus, Pdc2p can exert full transactivation activity by recruiting general transcription factors efficiently. It is likely that Pdc2p binds the upstream region of THI genes, and Mojzita & Hohmann (2006) noted that Pdc2p actually binds DNA, although the experimental Screening Library clinical trial data were not published. In this paper, we demonstrated, using chromatin immunoprecipitation (ChIP) assays, that Pdc2p interacts with the upstream region of THI genes, the sequences of which are different from the target sequence of Thi2p. It was also found that Pdc2p interacts with PDC5. Interestingly, the association of Pdc2p or Thi2p with the target DNA sequences of THI genes was enhanced by thiamin starvation, whereas the association of Pdc2p with the PDC5 promoter was unaffected. Furthermore, we identified a DNA element in the upstream region of
PDC5, which can bind to Pdc2p and is required for the expression of PDC5. The TA-cloning vector pGEM® T-Easy (Promega) was used to clone PDC2 gene and the PDC5 promoter isolated from yeast genomic DNA by PCR using Ex Taq™ DNA polymerase (Takara Bio, Otsu, Japan) with specific primers. The expression vectors are listed in Table 1. In general, the target sequence was PCR-amplified from the vector pGEM-PDC2 or pGEM-PDC5-promoter ADAMTS5 using specific primers into which restriction sites were designed, and the fragment obtained was digested with the restriction enzymes and subcloned into expression vectors. The PDC5 promoter-lacZ plasmids (B593ΔX series) carried an in-frame fusion between the inserted promoter-associated start codon and the lacZ coding sequence. All PCR primers are available on request. Escherichia coli strains DH5α and BL21(DE3)pLysS were used to amplify plasmids and express the recombinant proteins, respectively. Saccharomyces cerevisiae strains YPH500 (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1-Δ63 ade2-101 lys2-801), NKC18 (thi3::HIS3 in YPH500), and NKC19 (thi2::HIS3 in YPH500) (Nosaka et al., 2005) were used in this study.