To probe further the role of the Helix 5 tyrosine in the p270 ARID, the Y1096A substitution was combined with the P1042A substitution. The effect of the combined mutations was highly synergistic (Figure 11), generating a DNA-binding profile almost as defective as that seen with the W1073A substitution. This confirms that the Helix 5 tyrosine is important to structural integrity in the p270 ARID, but the results suggest that the p270 ARID is more able than the Dri ARID to tolerate changes in its aromatic scaffold. Thus, there appear to be fundamental differences in the ARID structures of p270 and Dri that go beyond simple differences at specific amino acid positions. This is consistent with the detrimental effects observed above of exchanging presumably analogous sequences between the two proteins. The mutagenesis studies argue against a conclusion that specific amino acids in Loop 2 or Helix 5 are the main determinants of sequence specificity. Most probably, this is determined by multiple interacting differences across the entire ARID structure.
