Identification of preferred binding sites of a light-inducible DNA-binding factor (MNF1) within 5'-upstream sequence of C4-type phosphoenolpyruvate carboxylase gene in maize

Abstract

MNF1 is a factor which specifically binds to a 318 bp fragment (-1012 to -695) in the 5'-flanking region of the C4-type phosphoenolpyruvate carboxylase gene in Zea mays (Yanagisawa et al., Mol Gen Genet 224 (1990) 325-332). The most preferred binding site of MNF1 determined by a 2 bp mutation-scanning assay was an octamer sequence, GTGCCCTT, which is located within the repeated sequences (RS1; -886 to -849, -846 to -807). Furthermore, a PCR-mediated selection-amplification assay identified both the octamer sequence, GTGCCC(A/T)(A/T), and an additional sequence. CC(G/A)CCC, the latter of which was similar to the Sp1 sites in vertebrates. Specific binding of MNF1 to each of the supposed binding sites was confirmed with double-stranded monomers as probes. Considering native molecular mass of MNF1 (ca. 500 kDa), a protein complex is expected. In addition, MNF1 is anticipated to have two distinct DNA-binding proteins since the MNF1 binding to CCGCCC element was 1,10-phenanthroline-dependent whereas the MNF1 binding to the octamer was independent. Wide distribution of the MNF1 binding sequences within the 1 kb promoter region accounts for broad interactions of MNF1. Moreover, specific DNA binding due to MNF1, which was not observed in the nuclear extract derived from germinated and cultivated plants in darkness, appeared after a white-light pulse. This finding suggests the involvement of the protein complex in the light-dependent transcriptional control in the gene expression.