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The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 μs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA., Competing Interests: Competing interests statement:The authors declare no competing interest.

A novel benzo[a]phenoxazine-based fluorescent dye LV2 has been employed as a molecular reporter to probe recognition of a linker histone protein H1 by calf-thymus DNA (DNA). Fluorescence lifetime of LV2 buried in the globular domain of H1 (∼2.1 ns) or in the minor groove of DNA (∼0.93 ns) increases significantly to 2.65 ns upon interaction of the cationic protein with DNA indicating formation of the H1-DNA complex. The rotational relaxation time of the fluorophore buried in the globular domain of H1 increases significantly from 2.2 ns to 8.54 ns in the presence of DNA manifesting the recognition of H1 by DNA leading to formation of the H1-DNA complex. Molecular docking and molecular dynamics (MD) simulations have shown that binding of LV2 is energetically most favourable in the interface of the H1-DNA complex than in the globular domain of H1 or in the minor groove of DNA. As a consequence, orientational relaxation of the LV2 is significantly hindered in the protein-DNA interface compared to H1 or DNA giving rise to a much longer rotational relaxation time (8.54 ns) in the H1-DNA complex relative to that in pure H1 (2.2 ns) or DNA (5.7 ns). Thus, via a significant change of fluorescence lifetime and rotational relaxation time, the benzo[a]phenoxazine-based fluorescent dye buried within the globular domain of the cationic protein, or within the minor groove of DNA, reports on recognition of H1 by DNA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Complexes of cationic liposomes with DNA have emerged as promising nonviral vectors for delivering genetic information into cells for gene therapy. Kinetics of the liposome/DNA complex (lipoplex) formation on a millisecond time scale are studied by monitoring time evolution of fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) and ethidium bromide (EtBr) in a continuous flow microfluidic channel coupled to a fluorescence microscope. The formation of lipoplexes between calf thymus DNA and liposomes based on two novel cationic lipids (Lip1810 and Lip1814) are found to follow a two-step process with kinetic constants for the Lip1814/DNA complex ( k 1 = 1120-1383 s -1 , k 2 = 0.227-1.45 s -1 ) being significantly different from those ( k 1 ) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is -1 whereas the formation of Lip1810/DNA complex occurs by a k . The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.2 = 32.3-60.19 s -1 ) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is diffusion-controlled whereas the formation of Lip1810/DNA complex occurs by a conformational rearrangement-controlled pathway . The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.

The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5'-GGGRNNNYCC-3' (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo., (Published by Oxford University Press on behalf of Nucleic Acids Research 2019.)

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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nuclear factor κB (NF-κB) regulates gene expression by binding to specific DNA elements, known collectively as κB sites, that are contained within the promoters/enhancers of target genes. We found that the identity of the central base pair (bp) of κB sites profoundly affects the transcriptional activity of NF-κB dimers. RelA dimers prefer an A/T bp at this position for optimal transcriptional activation (A/T-centric) and discriminate against G/C-centric κB sites. The p52 homodimer, in contrast, activates transcription from G/C-centric κB sites in complex with Bcl3 but represses transcription from the A/T-centric sites. The p52:Bcl3 complex binds to these two classes of κB sites in distinct modes, permitting the recruitment of coactivator, corepressor, or both coactivator and corepressor complexes in promoters that contain G/C-, A/T-, or both G/C- and A/T-centric sites. Therefore, through sensing of bp differences within κB sites, NF-κB dimers modulate biological programs by activating, repressing, and altering the expression of effector genes., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

A hallmark of the NF-kappaB transcription response to inflammatory cytokines is the remarkably rapid rate of robust activation and subsequent signal repression. Although the rapidity of postinduction repression is explained partly by the fact that the gene for IkappaBalpha is strongly induced by NF-kappaB, the newly synthesized IkappaBalpha still must enter the nucleus and compete for binding to NF-kappaB with the very large number of kappaB sites in the DNA. We present results from real-time binding kinetic experiments, demonstrating that IkappaBalpha increases the dissociation rate of NF-kappaB from the DNA in a highly efficient kinetic process. Analysis of various IkappaB mutant proteins shows that this process requires the C-terminal PEST sequence and the weakly folded fifth and sixth ankyrin repeats of IkappaBalpha. Mutational stabilization of these repeats reduces the efficiency with which IkappaBalpha enhances the dissociation rate.

The X-ray structure of the nuclear factor-kappaB (NF-kappaB) p52:RelB:kappaB DNA complex reveals a new recognition feature not previously seen in other NF-kappaB:kappaB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of kappaB sites. We found that the Arg 125-sensitive kappaB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein-induced kink observed in this complex, which used an AT-rich kappaB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non-AT-rich kappaB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of kappaB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in kappaB sites when compared with other NF-kappaB dimers.

We describe here the X-ray crystal structure of NF-kappaB p50/RelB heterodimer bound to a kappaB DNA. Although the global modes of subunit association and kappaB DNA recognition are similar to other NF-kappaB/DNA complexes, this complex reveals distinctive features not observed for non-RelB complexes. For example, Lys274 of RelB is removed from the protein-DNA interface whereas the corresponding residues in all other subunits make base-specific contacts. This mode of binding suggests that RelB may allow the recognition of more diverse kappaB sequences. Complementary surfaces on RelB and p50, as revealed by the crystal contacts, are highly suggestive of assembly of multiple p50/RelB heterodimers on tandem kappaB sites in solution. Consistent with this model our in vitro binding experiments reveal optimal assembly of two wild-type p50/RelB heterodimers on tandem HIV kappaB DNA with 2 bp spacing but not by a mutant heterodimer where one of the RelB packing surface is altered. We suggest that multiple NF-kappaB dimers assemble at diverse kappaB promoters through direct interactions utilizing unique protein-protein interaction surfaces.

The NF-kappaB family members p65 (RelA) and c-Rel recognize similar DNA sequences, yet the phenotypes of mutant mice suggest that these proteins regulate distinct sets of genes. Here we demonstrate that 46 unique residues within an 86-residue segment of the Rel homology region (RHR) of c-Rel are responsible for the c-Rel requirement for Il12b gene induction by lipopolysaccharide in bone marrow-derived macrophages. These same residues were responsible for the c-Rel requirement for Il12a induction in dendritic cells, and in both instances, no evidence of c-Rel-specific coactivator interactions was found. Although the residues of c-Rel and p65 that contact specific bases and the DNA backbone within nuclear factor-kappaB (NF-kappaB) recognition sequences are identical, homodimers of c-Rel and of a chimeric p65 protein containing the critical c-Rel residues bound with high affinity to a broader range of NF-kappaB recognition sequences than did wild-type p65 homodimers. These results demonstrate that the unique functions of closely related transcription factor family members can be dictated by differences in the range of DNA sequences recognized at high affinity, despite having similar binding site consensus sequences and DNA contact residues.

The dimeric NF-kappaB transcription factors regulate gene expression by recognizing specific DNA sequences located within the promoters of target genes. The DNA sequences, referred to as kappaB DNA, are divided into two broad classes. Class I kappaB DNA binds optimally to p50 and p52 NF-kappaB subunits, while class II kappaB DNAs are recognized specifically by the NF-kappaB subunits c-Rel and p65. We determined the X-ray crystal structure of a class II kappaB DNA sequence at 1.60 A resolution. This structure provides a detailed picture of kappaB DNA hydration, counter ion binding, and conformation in the absence of NF-kappaB binding partner. X-ray structures of both class I and class II kappaB DNA bound to NF-kappaB dimers were determined previously. Additionally, the NMR solution structure of a class I kappaB DNA is known. Comparison of the protein-bound and unbound kappaB DNA structures reveals that the free form of both classes approximates ideal B-form DNA more closely. Local geometries about specific DNA bases differ significantly upon binding to NF-kappaB. This is particularly evident at the 5'-GG/CC base-pairs; a signature of NF-kappaB specific DNA binding sequences. Differential phosphate group conformations, minor groove widths, buckle, twist, and tilt angles are observed between bound and unbound kappaB DNA. We observe that the presence of an extra G:C base-pair, 5'- to the GGA sequence in class I kappaB DNA, alters the geometry of the two internal G:C base-pairs within the GGGA tetranucleotide, which explains, at least in part, the structural basis for distinct NF-kappaB dimer recruitment by the two different classes of kappaB DNA. Together, these observations suggest that NF-kappaB dimers recognize specific structural features of kappaB DNA in order to make sequence-specific complexes.

The use of synthetic gene delivery systems in human gene transfer is hampered by poor transfection efficiencies, largely because of the inability of DNA to translocate across the nuclear pore complex. A means to overcome this barrier is to bind the DNA to nuclear localization signals (NLSs), which are recognized by shuttling receptors of the nuclear import machinery. Here, we studied the intracellular transport of plasmid DNA microinjected into HeLa cell cytoplasm, alone or as a complex with intact or NLS-deleted NFkappaB p50, using confocal microscopy imaging. We found that association of NLS-carrying p50 with DNA facilitated not only nuclear entry of the DNA but also its migration through the cytoplasm toward the nucleus. Facilitated transport of p50-DNA complexes in the cytoplasm proceeded along microtubules in a dynein-dependent manner and is mediated by the heterodimeric nuclear transport receptor that recognizes the p50-born NLS.

The avian Rev-T retrovirus encodes the oncoprotein v-Rel, a member of the Rel/nuclear factor (NF)-kappaB transcription factor family. The aggressive oncogenic potential of v-Rel has arisen from multiple mutations within the coding sequence of the avian cellular protein c-Rel. In this study, using quantitative biochemical experiments, we have tested the role of a limited set of alterations between v-Rel and c-Rel located within the Rel homology region (RHR) of the family that might confer functional differences. Our results show that only a set of six mutations within the RHR of v-Rel are responsible for its ability to bind to a broad spectrum of kappaB-DNA that are normally regulated by distinct NF-kappaB dimers. We also observe that both v-Rel homodimer and p50/v-Rel heterodimer bind IkappaBalpha weakly compared to other cellular Rel/NF-kappaB dimers with transcription activation potential. We suggest that the ability of v-Rel homodimer to deregulate subunit-specific gene expression and its ability to evade IkappaB inhibition are crucial to its strong oncogenic potential.

During the past two decades, structural and biophysical studies of DNA-protein and RNA-protein complexes have enhanced our understanding of the physico-chemical basis of nucleic acid recognition by proteins. However, it remains unclear what protein surface features are most important for nucleic acid binding and whether the same protein surface could bind specifically to both DNA and RNA. The recently described X-ray crystal structure of the transcription factor NF-kappaB p50 homodimer bound to a high-affinity RNA aptamer allows the direct comparison of NF-kappaB-RNA and NF-kappaB-DNA binding modes. The RNA aptamer, which bears no sequence homology to natural NF-kappaB DNA targets, adopts a structure with similar physico-chemical properties to kappaB DNA and contacts a common nucleic-acid-binding 'consensus surface' on the p50 homodimer.

The DNA binding of three different NF-kappaB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-kappaB p50/p65 heterodimer is shown here to bind the kappaB DNA target site of the immunoglobulin kappa enhancer (Ig-kappaB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-kappaB complex. The heterodimer binds to the Ig-kappaB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-kappaB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 degrees C and 42 degrees C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-kappaB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.

Background: The NF-kappaB family of dimeric transcription factors regulates the expression of several genes by binding to a variety of related DNA sequences. One of these dimers, p65(RelA), regulates a subclass of these targets. We have shown previously that p65 binds to the 5'-GGAA T TTTC-3' sequence asymmetrically. In that complex one subunit base specifically interacts with the preferred 5' half site and the other subunit binds non-specifically to the 3' half site., Results: Here we describe the crystal structures of two new p65-DNA complexes. One complex contains a pseudosymmetric 5'-GGAA T TTCC-3' DNA sequence taken from the enhancer of the gene encoding interleukin 8 (IL-8) and the other contains the asymmetric 5'-GGAA T TCCC-3' target DNA taken from the enhancer of the gene encoding type VII collagen. As expected, the global positioning of the dimer on both DNA targets is roughly symmetric, however, the hydrogen-bonding patterns at the protein-DNA interfaces differ significantly. One of the p65 monomers in complex with the asymmetric DNA binds to an extra base pair located immediately upstream of the 5'-GGAA-3' half site. We also show that p65 binds to these targets with almost equal affinity and that different residues have variable roles in binding different kappaB targets., Conclusions: Taken together, these structures reveal that p65 exhibits the unique capability to specifically bind DNA targets of variable lengths from four to ten base pairs. Also, the small protein segment Arg41-Ser42-Ala43 is at least partially responsible for flexibility in DNA-binding modes.

Rel/NF-kappaB transcription factors form homo- and heterodimers with different DNA binding site specificities and DNA binding affinities. Several intracellular pathways evoked by a wide range of biological factors and environmental conditions can lead to the activation of Rel/NF-kappaB dimers by signaling degradation of the inhibitory IkappaB protein. In the nucleus Rel/NF-kappaB dimers modulate the expression of a variety of genes including those encoding cytokines, growth factors, acute phase response proteins, immunoreceptors, other transcription factors, cell adhesion molecules, viral proteins and regulators of apoptosis. The primary focus of this review is on structural and functional aspects of Rel/NF-kappaB:DNA complexes and their formation. The salient features of the Rel/NF-kappaB dimer:DNA structure are described, as are modes of transcriptional regulation by phosphorylation, altered DNA binding properties, varying protein conformations, and interactions with IkappaB proteins.

The NF-kappaB p50/p65 heterodimer is the classical member of the Rel family of transcription factors which regulate diverse cellular functions such as immune response, cell growth, and development. Other mammalian Rel family members, including the proteins p52, proto-oncoprotein c-Rel, and RelB, all have amino-terminal Rel-homology regions (RHRs). The RHR is responsible for the dimerization, DNA binding and cytosolic localization of these proteins by virtue of complex formation with inhibitor kappaB proteins. Signal-induced removal of kappaB inhibitors allows translocation of dimers to the cell nucleus and transcriptional regulation of kappaB DNA-containing genes. NF-kappaB specifically recognizes kappaB DNA elements with a consensus sequence of 5'-GGGRNYYYCC-3' (R is an unspecified purine; Y is an unspecified pyrimidine; and N is any nucleotide). Here we report the crystal structure at 2.9 A resolution of the p50/p65 heterodimer bound to the kappaB DNA of the intronic enhancer of the immunoglobulin light-chain gene. Our structure reveals a 5-base-pair 5' subsite for p50, and a 4-base-pair 3' subsite for p65. This structure indicates why the p50/p65 heterodimer interface is stronger than that of either homodimer. A comparison of this structure with those of other Rel dimers reveals that both subunits adopt variable conformations in a DNA-sequence-dependent manner. Our results explain the different behaviour of the p50/p65 heterodimer with heterologous promoters.

Background: Members of the rel/NFkappaB family of transcription factors play a vital role in the regulation of rapid cellular responses, such as those required to fight infection or react to cellular stress. Members of this family of proteins form homo- and heterodimers with differing affinities for dimerization. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Crystal structures of the rel/NFkappaB family members p50 and p65 in their DNA-bound homodimeric form have been solved. These structures showed that the residues from the dimerization domains of both p50 and p65 participate in DNA binding and that the DNA-protein and protein dimerization surfaces form one continuous overlapping interface. We desired to investigate the contribution of DNA to NFkappaB dimerization and to identify the mechanism for the selective association of rel/NFkappaB family peptides into transcriptionally active dimers., Results: We report here the crystal structures of the dimerization domains of murine p50 and p65 at 2.2 A and 2.0 A resolution, respectively. A comparison of these two structures suggests that conservative amino acid changes at three positions are responsible for the differences in their dimer interfaces. The presence of the target DNA does not change the dimer interface of either protein in any significant manner., Conclusions: These two structures suggest that the rel/NFkappaB family of transcription factors use only a few conservative changes in their amino acid sequences to form a host of dimers with varying affinities for dimerization. Amino acids at positions corresponding to 254, 267, and 307 of murine p50, function as primary determinants for the observed differences in dimerization affinity. The DNA-contacting charged amino acid sidechains from the dimerization domains are held in a similar conformation in both the DNA-bound and free states, therefore, no major structural rearrangement is required to bring these residues into contact with the DNA.

Archaea possess a basal transcriptional apparatus that resembles that of eukaryotes. Here we report the 2.1-A crystal structure of the archaeal transcription factor complex formed by the TATA-box-binding protein (TBP), the transcription factor IIB homolog, and a DNA target, all from the hyperthermophile Pyrococcus woesei. The overall fold of these two basal transcription factors is essentially the same as that of their eukaryotic counterparts. However, in comparison with the eukaryotic complexes, the archaeal TBP-DNA interface is more symmetrical, and in this structure the orientation of the preinitiation complex assembly on the promoter is inverted with respect to that seen in all crystal structures of comparable eukaryotic systems. This study of the structural details of an archaeal transcription factor complex presents the opportunity to examine the evolution of the basal eukaryotic transcriptional apparatus from a stereochemical viewpoint and to extend our understanding of the physical biochemistry of transcriptional initiation.

Four new studies of the assembly of different subsets of the preinitiation complex of RNA polymerase II have provided insight into how transcription factors interact with each other and with DNA. A heterotetramer of TBP-associated factors shows a histone-like fold.

The 2.3-A crystal structure of the transcription factor NK-kappa B p50 homodimer bound to a palindromic kappa B site reveals that the Rel homology region folds into two distinct domains, similar to those in the immunoglobulin superfamily. The p50 dimer envelopes an undistorted B-DNA helix, making specific contacts along the 10-base-pair kappa B recognition site mainly through loops connecting secondary structure elements in both domains. The carboxy-terminal domains form a dimerization interface between beta-sheets using residues that are strongly conserved in the Rel family.