High affinity binding of MEF-2C correlates with DNA bending.
To regulate lineage-specific gene expression in many cell types, members of the myocyte enhancer factor-2 (MEF-2) family of transcription factors cooperate with basic helix-loop-helix (bHLH) proteins, which show only limited intrinsic DNA binding specificity. We investigated the DNA binding properties of MEF-2C in vitro and show that the inherent bendability of the MEF site is one of the principal structural characteristics recognized by MEF-2C. Measurements of the apparent dissociation constants of MEF-2C complexes with several DNA sequences revealed that MEF-2C bound with high affinity to DNA sequences containing a MEF site. Mutations in the MEF site which did not affect the bendability of the DNA changed the free energy of binding only marginally. However, reducing the intrinsic bendability of the DNA binding site through an AA-->GC substitution increased the half-maximal binding concentration of MEF-2C by almost one order of magnitude. Electrophoretic mobility shift assays revealed markedly reduced MEF-2C binding to DNA containing 2,6-diaminopurine. On binding to MEF-2C the maximum ellipticity at 275 nm in the CD spectrum of DNA containing a MEF site was red shifted by 4 nm and its intensity reduced significantly, while a slight blue shift of <1 nm was observed for a mutant DNA sequence with reduced bendability (AA-->GC). Bending analysis by circular permutation assay revealed that the DNA in the cognate complex was bent by 49 degrees , while the DNA in the complex with the mutant oligonucleotide was largely unbent.
Meierhans
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D
Department of Chemistry, ETH-Zürich, Universitätstrasse 16, CH-8092 Zurich, Switzerland.
Sieber
M
M
Allemann
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RK
D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000378:metabolism; D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D064326:MEF2 Transcription Factors; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011687:Purines / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D011687:Purines; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D004247:DNA; D000225:Adenine; C030985:purine