Importance of a flanking AT-rich region in target site recognition by the 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 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 MIG1 site in order to elucidate its binding specificity. We found that only three mutations within the GC box retain the ability to bind MIG1: 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 MIG1 binding. This AT box is present 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 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 MIG1 bends DNA. We found that binding of MIG1 is associated with bending within the AT box. We conclude that DNA binding by a simple zinc finger protein such as MIG1 can involve both recognition of the GC box and flanking sequence preferences that may reflect local DNA bendability.
Lundin
M
M
Ludwig Institute for Cancer Research, Uppsala Biomedical Center, Sweden.
Nehlin
J O
JO
Ronne
H
H
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C085976:MIG1 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins