1. DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated
- Author
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Maria Carmen Mulero, Yidan Li, Vivien Ya-Fan Wang, H. Thien Nguyen, De-Bin Huang, Tapan Biswas, and Gourisankar Ghosh
- Subjects
Models, Molecular ,0301 basic medicine ,cooperativity ,Cooperativity ,Crystallography, X-Ray ,Medical and Health Sciences ,Biochemistry ,Mice ,chemistry.chemical_compound ,Transactivation ,0302 clinical medicine ,Models ,Transcription (biology) ,Promoter Regions, Genetic ,Crystallography ,dimerization ,RELA ,E-selectin ,Biological Sciences ,Cell biology ,Protein Structure and Folding ,E-Selectin ,Protein Binding ,Transcriptional Activation ,crystal structure ,Biochemistry & Molecular Biology ,kB site ,1.1 Normal biological development and functioning ,DNA transcription ,RelA ,Biology ,Promoter Regions ,03 medical and health sciences ,Genetic ,Protein Domains ,Underpinning research ,Genetics ,Animals ,Molecular Biology ,Transcription factor ,Binding Sites ,Base Sequence ,Transcription Factor RelA ,Molecular ,Promoter ,DNA ,Cell Biology ,Molecular biology ,DNA binding site ,030104 developmental biology ,Gene Expression Regulation ,chemistry ,Chemical Sciences ,X-Ray ,Generic health relevance ,Protein Multimerization ,030217 neurology & neurosurgery ,NF-kB transcription factor - Abstract
The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.
- Published
- 2017