GT-2: in vivo transcriptional activation activity and definition of novel twin DNA binding domains with reciprocal target sequence selectivity.
GT-2 is a novel DNA binding protein that interacts with a triplet functionally defined, positively acting GT-box motifs (GT1-bx, GT2-bx, and GT3-bx) in the rice phytochrome A gene (PHYA) promoter. Data from a transient transfection assay used here show that recombinant GT-2 enhanced transcription from both homologous and heterologous GT-box-containing promoters, thereby indicating that this protein can function as a transcriptional activator in vivo. Previously, we have shown that GT-2 contains separate DNA binding determinants in its N- and C-terminal halves, with binding site preferences for the GT3-bx and GT2-bx promoter motifs, respectively. Here, we demonstrate that the minimal DNA binding domains reside within dual 90-amino acid polypeptide segments encompassing duplicated sequences, termed trihelix regions, in each half of the molecule, plus 15 additional immediately adjacent amino acids downstream. These minimal binding domains retained considerable target sequence selectivity for the different GT-box motifs, but this selectivity was enhanced by a separate polypeptide segment farther downstream on the C-terminal side of each trihelix region. Therefore, the data indicate that the twin DNA binding domains of GT-2 each consist of a general GT-box recognition core with intrinsic differential binding activity toward closely related target motifs and a modified sequence conferring higher resolution reciprocal selectivity between these motifs.
Ni
M
M
Department of Plant Biology, University of California, Berkeley 94720, USA.
Dehesh
K
K
Tepperman
J M
JM
Quail
P H
PH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000235:genetics* / Q000378:metabolism*; D010834:Phytochrome / Q000096:biosynthesis / Q000235:genetics*; D051942:Phytochrome A; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011392:Proline; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014026:Nicotiana; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D010940:Plant Proteins
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010834:Phytochrome; D051942:Phytochrome A; D011392:Proline; D011994:Recombinant Proteins; D014157:Transcription Factors; C077393:rice nuclear protein GT-2; D010940:Plant Proteins