Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster tramtrack protein

Abstract

We show that the DNA-binding domain of the Drosophila melanogaster regulatory protein Tramtrack consists of a 66 amino acid sequence containing two zinc-finger motifs and a short sequence N-terminal to the first finger motif. This short N-terminal sequence is essential for DNA binding and we suggest it is involved in maintaining the threedimensional structure of the first finger domain, as has been seen in the nuclear magnetic resonance structure of one of the zinc-finger domains of the yeast transcription factor SWI5. The characterization of the DNA-binding activity of this 66 residue peptide (Δ911zf) shows that it binds in a sequence-specific manner, as a monomer, to a natural target site with an apparent K D $ ̃ 4 × 10 −7 m. The shortest Δ911zf binding site, which retains full affinity, consists of an 11 base-pair sequence with a one nucleotide overhang at each 5′ end. DNase I. hydroxyl radical and methylation protection footprinting studies show that, in common with other zinc-finger proteins, Δ911zf binds in the major groove of DNA. The data presented are consistent with the zinc-fingers of Tramtrack contacting both strands of the DNA, and thus the binding differs in detail to that observed in the crystal structure of the three zinc-fingers of Zif268 complexed to their target DNA.