ABSTRACT
Members of the MADS-box family of transcription factors are found in eukaryotes ranging from yeast to humans. In plants, MADS-box proteins regulate several developmental processes including flower, fruit and root development. We have investigated the DNA-binding mechanisms used by four such proteins in Antirrhinum majus, SQUA, PLE, DEF and GLO. SQUA differs from the characterised mammalian and yeast MADS-box proteins as it can efficiently bind two different classes of DNA-binding site. SQUA induces bending of these binding sites and the sequence of the site plays a role in determining the magnitude of these bends. Similarly, PLE and DEF/GLO induce DNA bending although the direction of the resulting bends differ. Finally, we demonstrate that the MADS-box and I-domains are sufficient for homodimer formation by SQUA. However, the K-box in SQUA can also act as an oligomerisation motif and in the full-length protein, the K-box plays a different role in mediating dimerisation in the context of SQUA homodimers or heterodimers with PLE. Together these results contribute significantly to our understanding of the function of SQUA and other plant MADS-box proteins at the molecular level.
Subject(s)
DNA, Plant/metabolism , DNA-Binding Proteins/metabolism , Plant Proteins/metabolism , Transcription Factors/metabolism , Binding Sites/genetics , DNA, Plant/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Dimerization , MADS Domain Proteins , Nucleic Acid Conformation , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Structure, Tertiary , Substrate Specificity/genetics , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent Km for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent Ki values of approximately 0.1 microM and 6 microM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (Ki(app) 54 microM). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 micrograms ml-1) or cytosol (350 micrograms cytosolic protein ml-1) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.
