RESUMO
DNA ring closure methods have been applied to TATA box DNA and its complex with the TATA box-binding protein (TBP). The J factors for cyclization (effective concentrations of one DNA end about the other) have been measured using cyclization kinetics, with and without bound TBP, for 18 DNA constructs containing the adenovirus major late promoter TATA box (TATAAAAG) separated by a variable helical phasing adapter from sequence-induced A-tract DNA bends. Six phasing lengths were used at three overall DNA lengths each. Cyclization kinetics were also measured in the absence of protein for the same set of molecules bearing a mutant TATA box (TACAAAAG). The results suggest that the TATA box DNA itself is strongly bent and anisotropically flexible, in a direction opposite to the bend induced by TBP, and that the mutant TACA box is much less bent/flexible. The bending and flexibility of the free DNA may govern the energetics of recognition of different DNA sequences by TBP, and the intrinsic bend may act to repress transcription complex assembly in the absence of TBP. The cyclization kinetics of TBP-DNA complexes in solution predict a geometry generally consistent with crystal structures, which show dramatic bending and unwinding. The novel observation of TBP-induced topoisomers suggests that this minicircle approach is able to distinguish TBP-induced unwinding from writhe (these cancel out in larger DNA), and this in turn suggests that changes in supercoiling in small topological domains can control TBP binding.
