Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins.
Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.
Maeo
K
K
Department of Cellular Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Japan. maeo@agr.nagoya-u.ac.jp
Hayashi
S
S
Kojima-Suzuki
H
H
Morikami
A
A
Nakamura
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors