Mechanisms of activation of interferon regulator factor 3: the role of C-terminal domain phosphorylation in IRF-3 dimerization and DNA binding

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-β 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 tandem-binding sites, which are twisted by ∼100° relative to each other, requires considerable work to untwist and/or bend the DNA

DNA-binding domain of GCN4 induces bending of both the ATF/CREB and AP-1 binding sites of DNA

The interaction of proteins with DNA results, in some cases, in DNA bending, and this might have functional importance. However, when the protein-induced bending of DNA is small, its measurement presents a problem. It is shown that the fluorescence resonance energy transfer between fluorophores placed on the ends of the specially designed U-shaped DNA, which contains the DNA-binding sites at its central part, can be successfully used for this purpose. The lever effect of the arms of such U-shaped DNA ensures that the distance between the fluorophores is very sensitive to bending of the central part. Using this technique, it was shown that (i) the AP-1 and ATF/CREB binding sites of GCN4 transcription factor are pre-bent to the same extent (∼12° toward the major groove) and (ii) binding of the GCN4 DNA-binding domain (GCN4-bZIP) results in additional bending of both these target sites but to a greater extent at the ATF/CREB site. In total, in the complex with GCN4-bZIP, the ATF/CREB site is bent by (25 ± 2)° and the AP-1 site by (20 ± 2)° toward the minor groove