The bifactorial endosperm box of γ-zein gene:: characterisation and function of the Pb3 and GZM cis-acting elements

The proximal region of the gamma-zein promoter (gamma Z) has a functional bifactorial prolamin box element containing two cis-acting elements, a prolamin-box motif (Pb3) and a GCN4-like motif (GZM). By particle bombardment of maize endosperms with 5' deletions and internal deletions of gamma Z fused to the GUS gene, we have shown that a 135 bp region containing the bifactorial element is involved in the transcriptional activation of the gamma Z promoter. However, the 135 bp region was unable to activate the gamma Z promoter in the absence of a 84 bp downstream sequence. Using in vivo footprinting and gel mobility shift assays with 15 DAP endosperm nuclear extracts, we have demonstrated the presence of trans-acting factors that interact with Pb3 and GZM target sites. Base-substitution mutations within Pb3 and GZM decreased transcription activity of the gamma Z promoter suggesting a co-ordinated function between the two cis-acting elements. Two additional cis-motifs upstream of the bifactorial prolamin element have been identified: a motif with high homology to the AACA elements of rice glutelin genes and an AZM motif containing an ACGT core which binds nuclear proteins other than the Opaque 2 (O2).