p270 Loop 2 does not appear to contact DNA directly (15), but to evaluate the possibility that Loop 2 affects the orientation of Helix 5 within the major groove, a p270 chimera was constructed in which the Loop 2 sequence of Dri was placed in the p270.TFT construct. This chimera, designated p270.L2.TFT, contains the major groove contact residues of Dri as well as a Loop 2 sequence derived entirely from Dri, which, therefore, should be sufficiently long and flexible to permit proper orientation of the DNA contact residues within the major groove. Nonetheless, when tested in the DNA pull-down assay, the p270.L2.TFT chimera shows no greater tendency to sequence specificity than wild-type p270, and, indeed, has slightly less overall affinity for DNA (Figure 7). Since the TFT substitution alone did not affect p270 DNA-binding affinity, it is likely that the introduction of the exogenous Loop 2 sequence created a distortion that interferes with the overall strength of DNA contact in the p270 ARID.
