Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element.

Abstract

By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the y-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter. The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization. A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells. In vitro binding and DNase I footprinting assays with nuclear proteins prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region. The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes. A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro. It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner. Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor. The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the a-class zein promoter. These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes.

Amplicons of maize zein genes are conserved within genic but expanded and constricted in intergenic regions

“…Although these genes are interdispersed with unrelated sequence fragments, they are organized in a relatively dense fashion with an average distance of 6 kb, assuming about 2 kb for each gene. While the gene size may not be precise, it has previously been shown with natural variants and promoter deletion analysis that zein promoters are not larger than 1 kb in size (Ueda et al, 1994). Since these genes do not have any introns, their transcribed region is also very small, with 0.9 kb in length.…”

The Development of Endosperm in Grasses

“…Accumulation of prolamins in the endosperm, however, is controlled primarily at the transcriptional level, and the proteins accumulate generally during middle and late periods of endosperm development, according to specific spatial/temporal patterns Shewry et al, 2003;Halford and Shewry, 2007;Xu and Messing, 2008). A critical regulatory sequence for prolamin gene expression was first identified in barley and termed the 2300 element; later, related sequences were found in wheat, rye, and maize prolamin promoters (Forde et al, 1985;Ueda et al, 1994). This highly conserved region typically contains two motifs: the prolamin box and a GCN4-like sequence (Halford and Shewry, 2007;Marzabal et al, 2008).…”

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

“…Further analysis of these protein-DNA interactions indicate that they may be specific to the endosperm (13,14). Transient expression assays of zein gene promoter activity in maize endosperm suspension culture cells (12,14) suggest that the P-box plays a positive role in the coordinate activation of zein gene expression during endosperm development. Interestingly, the P-box in the 22-kDa zein gene promoter lies just 20 nucleotides upstream of the binding site for O2, suggesting that O2 may interact with factors binding the P-box to activate 22-kDa zein gene expression (5).…”

“…The P-box was initially identified on the basis of both its highly conserved nucleotide sequence (5Ј-TGTAAAG-3Ј) and position (Ϫ300 region) relative to the translation start codon of prolamin genes (9,10). Endosperm nuclear factors have been shown to bind the P-box present in the 19-, 22-and 27-kDa zein gene promoters (11,12). Further analysis of these protein-DNA interactions indicate that they may be specific to the endosperm (13,14).…”