As no transcriptional regulator other than Pdc2p involved in the expression of PDC5 has been identified to date, it is possible that the sequence recognized by Pdc2p is located in the region between −418 and −346. We, therefore, carried out an EMSA to determine whether Pdc2p can bind to this restricted region. Several double-stranded oligonucleotides

30–40 bp in length were designed Belnacasan purchase from the above region and used as the DNA probes (Table S2). The recombinant Pdc2p(1–581) purified as a histidine-tagged protein from E. coli cells (Fig. S1) was used in this experiment, as full-length Pdc2p and Pdc2p(1–406) were not expressed in E. coli cells. As a result, when the probe #5–1 corresponding to the region from −410 to −379 was mixed with Pdc2p(1–581), a band migrating

more slowly than the free probe was detected (Fig. 3b). In addition, the DNA probe in which one nucleotide was deleted from the 3′- or 5′- side of #5–1 did not confer retardation (data not shown). This shifted band was depleted by competition with a 125-fold molar excess of unlabeled #5–1, suggesting that Pdc2p can specifically bind to this sequence. It is likely that this sequence acts as a cis-acting element indispensable for PDC5 expression. Furthermore, we noticed that a DNA sequence partially homologous to #5–1 was located immediately SRT1720 clinical trial upstream of the Thi2p-recognition site of PHO3 (Fig. 3a). Then, to determine whether

Pdc2p also recognizes this homologous sequence, several oligonucleotides were prepared for an EMSA. selleck chemical As shown in Fig. 3, unlabeled #3–2 (corresponding to the region from −273 to −234), and to a lesser extent #3–1 (−256 to −227), were found to partly compete with #5–1 for binding to Pdc2p(1–581). Nevertheless, no shifted bands appeared when #3–1, #3–2, and their elongated oligonucleotides were used as labeled probes (data not shown), suggesting that the interaction between Pdc2p and the PHO3 upstream region is not strong enough to be detected under our in vitro assay conditions. We have previously demonstrated that, in addition to the region from −234 to −215 containing the Thi2p-recognition site, the deletion of −273 to −245 in the PHO3 promoter causes the decrease in expression (Nosaka et al., 1992). Pdc2p can probably bind to the region from −273 to −234 and transactivate the PHO3 gene together with Thi2p, which binds the closely spaced site. Until now, we could not identify the Pdc2p-recognition site in the THI4 and THI20 promoters by EMSA using oligonucleotides with a sequence similar to #5–1 or #3–2. From these findings, we assume that Pdc2p binds the recognition sites of THI genes with low affinity, and therefore, the presence of Thi2p with Thi3p is required for the satisfactory recruitment of Pdc2p to THI promoters.