The role of the phosphorus BI–BII transition in protein–DNA recognition: the NF-κB complex

We examined, by (1)H and (31)P NMR, the solution structure of a 16 bp non-palindromic DNA fragment (16M2) containing the HIV-1 NF-κB-binding site, in which the sequences flanking the κB site had been mutated. (31)P NMR was particularly useful for obtaining structural information on the phosphodiester backbone conformation. Structural features were then compared with those of the two previously studied DNA fragments corresponding, respectively, to the native κB fragment (16N) and a fragment in which mutations have been introduced at the 5′ end of the κB site (16M1). For the mutated 16M2 duplex, NMR data showed that the BI–BII equilibrium, previously reported for the native fragment (16N) at the κB flanking steps, was lost. The role of the BI–BII equilibrium in NF-κB recognition by DNA was then investigated by electrophoretic mobility shift assay. We found that the isolated κB site has the potential to bind efficiently due to the BI–BII equilibrium of the κB flanking sequences.