IMPORTANCE OF A FLANKING AT-RICH REGION IN TARGET SITE RECOGNITION BYTHE GC BOX-BINDING ZINC-FINGER PROTEIN MIG1

MIG1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. MIG1 is related to the mammalian Krox/ Egr, Wilms' tumor, and Spl finger proteins. It has two fingers and bin ds to a GCGGGG motif that resembles the GC boxes recognized by these m ammalian proteins. We have performed a complete saturation mutagenesis of a natural MIG1 site in order to elucidate its binding specificity. We found that only three mutations within the GC box retain the abili ty to bind MTG1: G(1) to C, C-2 to T, and G(5) to A. This result is co nsistent,vith current models for zinc finger-DNA binding, which assume that the sequence specificity is determined by base triplet recogniti on within the GC box. Surprisingly, we found that an AT-rich region 5' to the GC box also is important for MIG1 binding. This AT box is pres ent in all natural MIG1 sites, and it is protected by MIG1 in DNase I footprints. However, the AT box differs from the GC box in that no sin gle base within it is essential for binding. Instead, the AT-rich natu re of this sequence seems to be crucial. The fact that AT-rich sequenc es are known to increase DNA flexibility prompted us to test whether M IG1 bends DNA. We found that binding of MIG1 is associated with bendin g within the AT box. We conclude that DNA binding by a simple zinc fin ger protein such as MIG1 can involve both recognition of the GC box an d flanking sequence preferences that may reflect local DNA bendability .