Isolation of carrot basic leucine zipper transcription factor using yeast one-hybrid screening

Abscisic acid (ABA) is involved in various physiological and developmental processes, including stress responses and seed maturation. Many ABA-regulated genes associated with these processes have been identified and analyzed. Previously, we identified 2 important elements in the promoter of the carrotDcECP31 gene: motif X (CACACGTGGG), which is like an ABA-responsive element (ABRE), and motif Y (CACACGTATC). Together, these are sufficient for embryo-specific ABA-inducible promoter activity. We also showed that motif X functions is an enhancerlike element and that motif Y participates in ABA responsiveness. In this study, we isolated the nuclear protein that interacts with motif Y of theDcECP31 promoter. We performed yeast one-hybrid screening using integrated motif Y as bait and isolated clones. Sequence analysis revealed that clone 22 included the carboxyl-terminal half of bZIP, which contains the basic and leucine zipper domains and binds to G-boxes containing the sequence ACGT. This result supports the hypothesis that carrot C-ABI3, a homologue of theArabidopsis ABI3 protein, functions as a coactivator that interacts with the G-box via protein-protein contacts and suggests that the complex controls the expression of theDcECP31 gene.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.