The basal control region (BCR) and CSRE_LAC4 regulate transcription of the LAC4 gene.

(A) Intergenic region between the divergently transcribed genes LAC12 and LAC4 containing the basal control region (BCR, shaded in dark gray) and CSRE_LAC4 (black box). Four binding sites for the transcription activator KlGal4 are indicated by light grey boxes. Distances are given relative to the LAC4 ATG. (B) Non-induced LAC4 mRNA levels in wild-type and a BCR deletion mutant (ΔBCR; strain JA6/LR2). Total RNA was isolated from cells grown in 3% glycerol and analyzed by quantitative S1 nuclease mapping as described previously [39]. Histone H3 RNA (HHT1, lower lanes) served as loading control.(C) Transcription activation function of CSRE_LAC4. Non-induced LAC4 expression was determined by β-galactosidase measurements in extracts from wild-type (JA6), JA6/LR2 (ΔBCR) and JA6/LR2K (ΔBCR::CSRE_LAC4) strains grown in synthetic complete medium containing 2% glucose, 2% acetate or 2% ethanol. (D) Dependence of CSRE_LAC4–protein complex formation on KlCAT8 and KlSIP4. 10, 20 or 40 μg of S100 protein extracts from wild-type (JA6), sip4Δ (JA6/DS4), cat8Δ (yIG8) or sip4Δcat8Δ (yIG8/DS4) cells grown in synthetic complete medium with 3% glycerol were used for EMSA with a labeled CSRE_LAC4 oligonucleotide (~80,000 cpm).