Importance of a Flanking AT-Rich Region in Target Site Recognition by the GC Box-Binding Zinc Finger Protein MIGl

Abstract

MIGl is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. MIGl is related to the mammalian Krox/Egr, Wilms’ tumor, and Sp1 finger proteins. It has two fingers and binds to a GCGGGG motif that resembles the GC boxes recognized by these mammalian proteins. We have performed a complete saturation mutagenesis of a natural MIGl site in order to elucidate its binding specificity. We found that only three mutations within the GC box retain the ability to bind MIGl: G₁ to C, C₂ to T, and G₅ to A. This result is consistent with current models for zinc finger-DNA binding, which assume that the sequence specificity is determined by base triplet recognition within the GC box. Surprisingly, we found that an AT-rich region 5′ to the GC box also is important for MIGl binding. This AT box is present in all natural MIGl sites, and it is protected by MIGl in DNase I footprints. However, the AT box differs from the GC box in that no single base within it is essential for binding. Instead, the AT-rich nature of this sequence seems to be crucial. The fact that AT-rich sequences are known to increase DNA flexibility prompted us to test whether MIGl bends DNA. We found that binding of MIGl is associated with bending within the AT box. We conclude that DNA binding by a simple zinc finger protein such as MIGl can involve both recognition of the GC box and flanking sequence preferences that may reflect local DNA bendability.