The ARID is categorized as a modified helix–turn–helix motif-based DNA-binding domain, in which the second helix of the motif (Helix 5) is the recognition helix. To test the possibility that the first helix of the motif (Helix 4) influences the orientation of Loop 2 and the recognition helix, the p270.L2.TFT construct was further modified such that the entire region from the beginning of Helix 4 to the last major groove contact residue consists of contiguous Dri sequence. The name of this mutant is p270.H4.L2.TFT. This construct still binds to DNA without any clear sequence selectivity (Figure 7). Moreover, affinity is reduced below that of the p270.L2.TFT variant, supporting the suggestion above, that introduction of exogenous sequence creates distortions that interfere with the overall strength of DNA contact in the p270 ARID. Individual elements are not directly exchangeable between different ARIDs. Together, these results indicate that sequence specificity in the ARID does not depend solely on the specific amino acid composition in the major groove contact region.
