FIGURE 5.

Redox regulation of AtbZIP16 and its DNA binding activity. A, the quaternary structure of AtbZIP16 protein was analyzed under reducing and oxidizing conditions by incubating AtbZIP16 in the presence of either 0–10 mm DTT or 0–50 mm H₂O₂. Proteins were detected using anti-His antibody, and positions of monomers, dimers, and oligomers are indicated by arrows. B, the effect of DTT and H₂O₂ on DNA binding activity of AtbZIP16 was analyzed by EMSA. Binding reactions were carried out in the absence or presence of AtbZIP16, 10 mm DTT, and 10 mm H₂O₂. Positions of free DNA and protein-DNA complexes are indicated by arrows. C, intermolecular disulfide bond formation in AtbZIP16. AtbZIP16 protein or its mutant variants were either reduced with β-mercaptoethanol (β-ME) or left in their unreduced forms before loading for non-reducing SDS-PAGE. Locations of the monomers, dimers, and oligomers are indicated by arrows for AtbZIP16-WT protein, AtbZIP16 containing a Cys to Leu mutation at position 330, AtbZIP16 containing a Cys to Leu mutation at position 358, and AtbZIP16 containing Cys to Leu mutations at positions 330 and 358. D, binding of AtbZIP16-WT protein and its mutant variants to the G-box element. EMSA was performed using the biotin-labeled G-box element without protein or with AtbZIP16-WT, AtbZIP16 containing a Cys to Leu mutation at position 358, AtbZIP16 containing a Cys to Leu mutation at position 330, and AtbZIP16 containing Cys to Leu mutations at positions 330 and 358. Positions of free DNA and protein-DNA complexes are indicated by arrows. E, Coomassie staining of purified His-tagged bZIP16 protein containing only the basic region and lacking the leucine zipper (bZIP16BR). The band corresponding to the respective protein is indicated by an arrow. bZIP16BR was analyzed for the formation of DNA-protein complex with G-box by EMSA. Binding reactions contained the biotin-labeled G-box element in the absence or presence of AtbZIP16BR. Positions of free DNA and protein-DNA complexes are indicated by arrows.