Nucleic Acids Research, Vol 25, Issue 22 4537-4544, Copyright © 1997 by Oxford University Press
D Meierhans, M Sieber and RK Allemann
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.
ARTICLES
High affinity binding of MEF-2C correlates with DNA bending
Department of Chemistry, ETH-Zurich, Universitatstrasse 16, CH-8092 Zurich, Switzerland.
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