Base sequence discrimination by zinc-finger DNA-binding domains

ZINC fingers1,2 constitute important eukaryotic DNA-binding domains, being present in many transcription factors3–5. The Cys2/His2zinc-finger class has conserved motifs of 28–30 amino acids which are usually present as tandem repeats1,2. The structure of a Cys2/His2zinc finger has been determined by nuclear magnetic resonance6, but details of its interaction with DNA were not established. Here we identify amino acids governing DNA-binding specificity using in vitro directed mutagenesis guided by similarities between the zinc fingers of transcription factors Spl (ref. 7) and Krox-20 (ref. 8). Krox-20 is a serum-inducible transcription activator8,9 which is possibly involved in the regulation of hindbrain development10; it contains three zinc fingers similar to those of Spl (refs 7, 8, 11) and binds to a 9-base-pair target sequence which is related to that of Spl (ref. 9). Our results show that each finger spans three nucleotides and indicate two positions in Krox-20 zinc fingers that are important for base-pair selectivity. Modelling with molecular graphics suggests that these residues could bind directly with the bases and that other amino acid–base contacts are also possible.