Monitoring the interaction between DNA and a transcription factor (MEF2A) using fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) is an analytical method that allows distinguishing different populations of fluorescent probes in solution and provides data on their concentrations and their diffusion coefficients. FCS was used to characterize the interaction of the transcription factor (MEF2A) with its DNA target sequence. The myocyte enhancer factor 2 (MEF2) belongs to the MADS-box family and activates transcription of numerous muscle genes during myogenesis. Measurements were made using TAMRA-labelled oligonucleotide duplexes derived from a wild type (WT) or a mutated MEF2 target gene. Binding of the protein to the WT DNA resulted in significant changes of the diffusion. Specificity of the interaction was confirmed using the mutated DNA. Bound to free probe ratios were determined at different MEF2A concentrations and the apparent equilibrium dissociation constant K(D) for the full-length MEF2A was estimated.
Octobre
Guillaume
G
Inserm U309, institut Albert-Bonniot, domaine de la Merci, 38706 La Tronche cedex, France.
Lemercier
Claudie
C
Khochbin
Saadi
S
Robert-Nicoud
Michel
M
Souchier
Catherine
C
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D026161:MADS Domain Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; C578035:MEF2A protein, human; D018006:Myogenic Regulatory Factors; D011994:Recombinant Proteins; D004247:DNA