The Floral Homeotic Protein SEPALLATA3 Recognizes Target DNA Sequences By Shape Readout Involving A Conserved Arginine Residue In The MADS-Domain

SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor and a central player in flower development. MADS-domain proteins bind as dimers to AT-rich sequences termed CArG-boxes which share the consensus 5-CC(A/T)6GG-3. Since only a fraction of the abundant CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-box sequences into genuine recognition sites. Here, we investigated to which extent the shape of the DNA contributes to the DNA-binding specificity of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to a variety of DNA probes which all contain the CArG-box motif, but differ in their DNA shape characteristics. We found that binding affinity correlates well with certain DNA shape features associated with A-tracts. Analysis of SEPALLATA3 proteins with single amino acid substitutions in the DNA-binding MADS-domain further revealed that a highly conserved arginine residue, which is expected to contact the DNA minor groove, contributes significantly to the shape readout. Our studies show that the specific recognition of cis-regulatory elements by plant MADS-domain transcription factors heavily depend on shape readout mechanisms and that the absence of a critical arginine residue in the MADS-domain impairs binding specificity.