TBP flanking sequences: asymmetry of binding, long-range effects and consensus sequences

We carried out in vitro selection experiments to systematically probe the effects of TATA-box flanking sequences on its interaction with the TATA-box binding protein (TBP). This study validates our previous hypothesis that the effect of the flanking sequences on TBP/TATA-box interactions is much more significant when the TATA box has a context-dependent DNA structure. Several interesting observations, with implications for protein–DNA interactions in general, came out of this study. (i) Selected sequences are selection-method specific and TATA-box dependent. (ii) The variability in binding stability as a function of the flanking sequences for (T-A)(4) boxes is as large as the variability in binding stability as a function of the core TATA box itself. Thus, for (T-A)(4) boxes the flanking sequences completely dominate and determine the binding interaction. (iii) Binding stabilities of all but one of the individual selected sequences of the (T-A)(4)form is significantly higher than that of their mononucleotide-based consensus sequence. (iv) Even though the (T-A)(4) sequence is symmetric the flanking sequence pattern is asymmetric. We propose that the plasticity of (T-A)(n) sequences increases the number of conformationally distinct TATA boxes without the need to extent the TBP contact region beyond the eight-base-pair long TATA box.