Analysis of the DNA-binding properties of MyoD, myogenin, and E12 by fluorescence anisotropy.
MyoD and Myogenin are dominant myogenic regulatory factors (MRFs), which are involved in control of muscle-specific gene expression. The ubiquitously expressed E12 dimerizes with MyoD and Myogenin and has been shown to enhance their DNA-binding and transcriptional activities. In this study, fluorescence anisotropy assays have been used to determine the Gibb's free energy of dissociation (DeltaG) for MyoD, Myogenin, and E12 as homo- and heterodimers to the well-characterized myosin light chain enhancer (MLC), muscle creatine kinase (MCK) enhancer, and mutant thereof. The heterodimers of MyoD or Myogenin with E12 bound the MCK enhancer equally well (DeltaG = 21 kcal/mol). The homodimers varied dramatically in both MLC and MCK enhancer binding affinity. MyoD homodimer bound the MCK enhancer with the highest affinity (DeltaG = 19.6 kcal/mol) in comparison with the Myogenin homodimer-MCK interaction (DeltaG = 16.6 kcal/mol) and E12 homodimer-MCK interaction (DeltaG = 18.0 kcal/mol). The slope and shape of the binding isotherms revealed that with the exception of the E12 homodimer-MCK enhancer interaction, the other proteins bound with high levels of positive cooperativity. In contrast, the E12 homodimer-MCK enhancer interaction actually occurs with significant negative cooperativity. The binding of these proteins to MLC enhancer mimicked binding to the MCK enhancer, but with much lower affinities. These data support the hypothesis that DNA acts as an allosteric ligand facilitating the dimerization of these proteins. The combination of differential affinity and cooperativity explains why the heterodimers are the active species in transcriptional regulation.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205, USA. sjmaleki@srrc.ars.usda.gov
Soheila J
SJ
Maleki
Catherine A
CA
Royer
Barry K
BK
Hurlburt
D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D024721:E-Box Elements / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017570:MyoD Protein / Q000737:chemistry* / Q000378:metabolism; D018008:Myogenin / Q000737:chemistry* / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D013050:Spectrometry, Fluorescence; D051820:TCF Transcription Factors; D013816:Thermodynamics; D017186:Titrimetry; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D017570:MyoD Protein; D018008:Myogenin; D018994:Myosin Light Chains; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D003402:Creatine Kinase