Fig. 1.

CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. (A) The BAS1 promoter harbors both EE and CBS motifs, while only one CBS motif exists in the SOB7 promoter. CCA1 interacted with pBAS1-EE (B) and pSOB7-CBS (C), but not with pBAS1-CBS1 (D) in targeted Y1H assays. CCA1 did not interact with pBAS1-EEm (E) or pSOB7-CBS (F), in which the EE or CBS motifs have been mutated, respectively. Interactions of CCA1 with pBAS1-EEm (G) and pSOB7-CBS (H) were confirmed by EMSA. For each Y1H interaction tested, the indicated bait sequence was integrated into the mutant HIS3 locus of the yeast strain YM4271. The bait-integrated yeast clone with the lowest self-activation was transformed with the indicated prey construct and empty prey vector (negative control), and then plated on selection medium supplemented with 3-AT at the concentrations shown. Yeast clones were grown at 28 °C for 3–4 d. Three independent clones were shown for each sample. For EMSA, His–CCA1 and MBP–CCA1 were incubated with pBAS1-EE and pSOB7-CBS, respectively, and separated by non-denaturing PAGE. DNA probes and protein–DNA complexes were stained by SYBR Green. MBP was used as a negative control.