Assembling the human IFN-beta enhanceosome in solution.
Assembly of interferon-beta enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII-PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-kappaB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-kappaB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-beta enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-kappaB binding affinity for the major groove of this site.
Dragan
A I
AI
Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
Carrillo
R
R
Gerasimova
T I
TI
Privalov
P L
PL
D051698:Activating Transcription Factor 2 / Q000378:metabolism; D016880:Anisotropy; D020029:Base Pairing; D001483:Base Sequence; D002151:Calorimetry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005453:Fluorescence; D024742:HMG-Box Domains; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism*; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012996:Solutions
D051698:Activating Transcription Factor 2; D003432:Cross-Linking Reagents; D050838:Interferon Regulatory Factor-3; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D012996:Solutions; D016899:Interferon-beta; D004247:DNA