Base sequence discrimination by zinc-finger DNA-binding domains

Abstract

Zinc fingers constitute important eukaryotic DNA-binding domains, being present in many transcription factors. The Cys2/His2 zinc-finger class has conserved motifs of 28-30 amino acids which are usually present as tandem repeats. The structure of a Cys2/His2 zinc finger has been determined by nuclear magnetic resonance, 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 Sp1 and Krox-20. Krox-20 is a serum-inducible transcription activator which is possibly involved in the regulation of hindbrain development; it contains three zinc fingers similar to those of Sp1 and binds to a 9-base-pair target sequence which is related to that of Sp1. 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.