Identification of a transcriptional activator-binding element in the 27-kilodalton zein promoter, the -300 element.
By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-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 protein 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 alpha-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.
Ueda
T
T
Waksman Institute, Rutgers University, Piscataway, New Jersey 08855-0759.
Wang
Z
Z
Pham
N
N
Messing
J
J
D001483:Base Sequence; D001665:Binding Sites; D017796:Caulimovirus / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003850:Deoxyribonuclease I; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism; D015028:Zein / Q000096:biosynthesis / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D015028:Zein; D003850:Deoxyribonuclease I