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 pol peptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, s equence similarity was basically limited to the two WRKY domains. Glutathio ne S-transferase fusion proteins with the C-terminal WRKY domain of four Nt WRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY dom ain showed weaker binding activity with the W-box compared to the C-termina l domain. The DNA-binding activity of the WRKY domain was abolished by o-ph enanthroline and this inhibition was recovered specifically by Zn2-. Substi tution of the conserved cysteine and histidine residues of the plant-specif ic C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA bi nding. In addition, mutations in the invariable WRKYGQK sequence at the N-t erminal side of the zinc finger-like motif also significantly reduced the D NA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.