Stability and DNA-Binding Ability of the bZIP Dimers Formed by the ATF-2 and c-Jun Transcription Factors (original) (raw)
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Nucleic acids research, 1991
The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two Bi and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the Bi complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OLl were recovered in B2. For each probe, the possibility of the part of the sequence involved in Bi complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secundary structure, the formal existence of a H-DNA structure has not been demonstrated.Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations.
Nucleic Acids Research, 1990
The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two Bi and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the Bi complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OLl were recovered in B2. For each probe, the possibility of the part of the sequence involved in Bi complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secundary structure, the formal existence of a H-DNA structure has not been demonstrated.Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations.
Proceedings of the National Academy of Sciences, 1997
Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences f lanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs f lanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs f lanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos-Jun-AP-1 complex may contribute to its functional versatility at different promoters.
Archives of Biochemistry and Biophysics, 2008
The Jun oncoprotein belongs to the AP1 family of transcription factors that is collectively engaged in diverse cellular processes by virtue of their ability to bind to the promoters of a wide spectrum of genes in a DNA sequence-dependent manner. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domain of Jun to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of Jun to both sites occurs with indistinguishable affinities but with distinct thermodynamic signatures comprised of favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures. Furthermore, anomalously large negative heat capacity changes observed provoke a model in which Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. Taken together, our data provide novel insights into the energetics of a key protein-DNA interaction pertinent to cellular signaling and cancer. Our study underscores the notion that the folding and dimerization of transcription factors upon association with DNA may be a more general mechanism employed in protein-DNA interactions and that the conventional school of thought may need to be reevaluated.
Biochemical and Biophysical Research Communications, 2010
Leucine zippers, structural motifs typically comprised of five successive heptads of amino acids with a signature leucine at every seventh position, play a central role in the dimerization of bZIP family of transcription factors and their subsequent binding to the DNA promoter regions of target genes. Herein, using analytical laser scattering (ALS) in combination with isothermal titration calorimetry (ITC), we study the effect of successive C-terminal truncation of leucine zippers on the dimerization and energetics of binding of bZIP domains of Jun transcription factor to its DNA response element. Our data show that all five heptads are critical for the dimerization of bZIP domains and that the successive C-terminal truncation of residues leading up to each signature leucine significantly compromises the binding of bZIP domains to DNA. Taken together, our study provides novel insights into the energetic contributions of leucine zippers to the binding of bZIP domains of Jun transcription factor to DNA.
Archives of Biochemistry and Biophysics, 2008
In response to mitogenic stimuli, the heterodimeric transcription factor Jun-Fos binds to the promoters of a diverse array of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryogenic development and cancer. In so doing, Jun-Fos heterodimer regulates gene expression central to physiology and pathology of the cell in a specific and timely manner. Here, using the technique of isothermal titration calorimetry (ITC), we report detailed thermodynamics of the bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of the bZIP domains to both TRE and CRE is under enthalpic control and accompanied by entropic penalty at physiological temperatures. Although the bZIP domains bind to both TRE and CRE with very similar affinities, the enthalpic contributions to the free energy of binding to CRE are more favorable than TRE, while the entropic penalty to the free energy of binding to TRE is smaller than CRE. Despite such differences in their thermodynamic signatures, enthalpy and entropy of binding of the bZIP domains to both TRE and CRE are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the free energy of binding. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change determined is much larger than that expected from the direct association of bZIP domains with DNA. This observation is interpreted to suggest that the basic regions in the bZIP domains are largely unstructured in the absence of DNA and only become structured upon interaction with DNA in a coupled folding and binding manner. Our new findings are rationalized in the context of 3D structural models of bZIP domains of Jun-Fos heterodimer in complex with dsDNA oligos containing the TRE and CRE consensus sequences. Taken together, our study demonstrates that enthalpy is the major driving force for a key protein-DNA interaction pertinent to cellular signaling and that protein-DNA interactions with similar binding affinities may be accompanied by differential thermodynamic signatures. Our data corroborate the notion that the DNA-induced protein structural changes are a general feature of the bZIP family of transcription factors.
Nucleic Acids Research, 2007
The interferon regulatory transcription factor (IRF-3) is activated by phosphorylation of Ser/Thr residues clustered in its C-terminal domain. Phosphorylation of these residues, which increases the negative charge of IRF-3, results in its dimerization and association with DNA, despite the increase in repulsive electrostatic interactions. To investigate this surprising effect, the dimerization of IRF-3 and two phosphomimetic mutants, 2D (S396D, S398D) and 5D (S396D, S398D, S402D, T404D and S405D), and their binding to single-site PRDI and double-site PRDIII-PRDI DNA sequences from the IFN-b enhancer have been studied. It was found that: (a) the mutations in the C-terminal domain do not affect the state of the DNA-binding N-terminal domain or its ability to bind target DNA; (b) in the 5D-mutant, the local increase of negative charge in the C-terminal domain induces restructuring, resulting in the formation of a stable dimer; (c) dimerization of IRF-3 is the basis of its strong binding to PRDIII-PRDI sites since binding of 5D to the single PRDI site is similar to that of inactivated IRF-3. Analysis of the binding characteristics leads to the conclusion that binding of dimeric IRF-3 to the DNA with two tandembinding sites, which are twisted by $1008 relative to each other, requires considerable work to untwist and/or bend the DNA.
Structure of IRF-3 Bound to the PRDIII-I Regulatory Element of the Human Interferon-β Enhancer
Molecular Cell, 2007
Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the assembly of the mammalian interferon-β (IFN-β) enhanceosome. We present here the structure of IRF-3 DNA binding domain in complex with the complete PRDIII-I regulatory element of the human IFN-β enhancer. We show that four IRF-3 molecules bind in tandem to, variably spaced, consensus and nonconsensus IRF sites on the composite element. The ability of IRF-3 to bind these variable sites derives in part from two nonconserved arginines (Arg78 and Arg86) that partake in alternate protein-DNA contacts. We also show that the protein-DNA contacts are highly overlapped and that all four IRF sites are required for gene activation in vivo. In addition, we show that changing the nonconsensus IRF sites to consensus sites creates a more efficient enhancer in vivo. Together, the structure and accompanying biological data provide insights into the assembly of the IFN-β enhanceosome in mammals.
Long-range electrostatic interactions influence the orientation of fos-jun binding at AP-1 sites
Journal of Molecular Biology, 2001
Heterodimeric transcription regulatory proteins that bind palindromic DNA sequences can potentially bind their recognition sites in two opposite orientations. The orientation of transcription factor binding can control transcriptional activity by altering interactions with proteins that bind to adjacent regulatory elements. Fos-Jun heterodimers bind to AP-1 sites with different¯anking sequences in opposite orientations. A gel-based¯uorescence resonance energy transfer assay, gelFRET, was used to de®ne the mechanism whereby amino acid residues and nucleotide base-pairs outside the Fos-Jun-AP-1 contact interface determine the orientation of heterodimer binding. Exchange of three amino acid residues adjacent to the basic DNA contact regions between Fos and Jun reversed the binding orientation. The effects of these amino acid residues on the orientation of heterodimer binding depended on base-pairs¯anking the core AP-1 recognition sequence. Single amino acid and base-pair substitutions had parallel effects on DNA bending by Fos-Jun-AP-1 complexes and on heterodimer orientation. The binding orientation exhibited a close correspondence with both the difference in bending propensities of opposite sides of the AP-1 site as well as the difference in bending potentials of the Fos and Jun subunits of the heterodimer. The in¯uence of¯anking DNA sequences on heterodimer orientation was attenuated in the presence of high concentrations of multivalent cations. Base substitutions up to one helical turn from the center of the AP-1 site affected the binding orientation. Modi®cation of¯anking base-pairs with positively or negatively charged functional groups had opposite effects on the orientation of heterodimer binding. These changes in DNA charge had converse effects on the orientation preferences of heterodimers in which charged amino acid residues adjacent to the basic regions were exchanged between Fos and Jun. These results indicate that the orientation of heterodimer binding is determined primarily by minimization of the electrostatic free energy of the Fos-Jun-AP-1 complex. Consequently, long-range electrostatic interactions in¯uence the architecture of nucleoprotein complexes.