Opaque-2 Is a Transcriptional Activator That Recognizes a Specific Target Site in 22-kD Zein Genes

Abstract

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.

A Rice Functional Transcriptional Activator, RISBZ1, Responsible for Endosperm-specific Expression of Storage Protein Genes through GCN4 Motif

“…These results suggest a possibility that RISBZ1 functions as an activator of the glutelin genes. Similar expression pattern has been reported in maize O2 (14), wheat SPA (5), and barley Blz2 genes (19).…”

“…the number of exons and introns is conserved and individual introns occur at relative the same sites as those of the maize O2 (37,14), sorghum O2 (38), coix O2 (39), and barley Blz1 genes (18) (Fig. 3).…”

“…Maize Opaque-2 (O2) is an endosperm-specific transcription factor belonging to the basic leucine zipper (bZIP) family that has been shown to bind to the ACGT motif of maize 22-kDa ␣-zein promoter and activates transcription (14). It has been also reported to be involved in activation of endosperm-specific transcription of b32 ribosome-inactivating protein gene via a distinct cis-sequence (G(a/t)TGAPyPuTGPu) (15), thus indicating that the O2 has broad binding specificity.…”

RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway

“…Nuclear and cytosolic fractions were obtained based on methods described in ref. 26, except that dissected endosperms were used, the supernatant above the Percoll cushion enriched for cytosolic proteins also was collected and processed, and the lysis buffer contained 0.15 M NaCl to preserve protein complexes. Immunoblotting, immunoprecipitation, and phosphorylation assays will be described elsewhere (R.A.D., P.A.S., and B.A.L., unpublished data).…”

A 45‐bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm‐specific expression of the rice storage protein glutelin gene,GluA‐3

“…These indic~ ted that the GCN4 motif generally enhances the promoter attivity but that the combination of the two motifs confers el dosperm-specific gene expression. The GCN4 motif consists of the consensus seven nucleoti, les (TGAPuTCA) forming a palindromic structure, which is recognized by a bZIP type trans-acting factor [27,28]. It has bten reported that the GCN4 motif is implicated in some plant gene promoter activation in combination with other el,~ments (i.e.…”