Search

Summary: SEPALLATA3 of Arabidopsis thaliana is a MADS‐domain transcription factor (TF) and a key regulator of flower development. MADS‐domain proteins bind to sequences termed ‘CArG‐boxes’ [consensus 5′‐CC(A/T)6GG‐3′]. Because only a fraction of the CArG‐boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG‐boxes into genuine recognition sites. Here, we investigate to what extent the shape of the DNA is involved in a ‘shape readout’ that contributes to the binding of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to DNA probes that all contain the CArG‐box motif, but differ in their predicted DNA shape. We found that binding affinity correlates well with a narrow minor groove of the DNA. Substitution of canonical bases with non‐standard bases supports the hypothesis of minor groove shape readout by SEPALLATA3. Analysis of mutant SEPALLATA3 proteins 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 a plant MADS‐domain TF, and by inference probably also of other TFs of this type, heavily depends on shape readout mechanisms. [ABSTRACT FROM AUTHOR]

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124–245 fragment of serum response factor) with specific oligonucleotides from c-fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core-SRF labeled with bis-ANS showed that the protein is monomeric at low concentration (10−7 m). The titration curves for the fluorescence anisotropy of fluorescein-labeled oligonucleotide revealed that under equilibrium conditions, the core-SRF monomers were bound sequentially to SRE at very low concentration (10−9 m). Curve-fitting data showed also major differences between the wild-type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core-SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core-SRF was bound as a dimer to the wild-type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far-UV CD spectra indicated that the flexibility of core-SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein-labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA–protein interaction. [ABSTRACT FROM AUTHOR]