Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein.
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 three-dimensional 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 SW15. The characterization of the DNA-binding activity of this 66 residue peptide (delta 911zf) shows that it binds in a sequence-specific manner, as a monomer, to a natural target site with an apparent KD approximately 4 x 10(-7) M. The shortest delta 911zf 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, delta 911zf 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.
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MRC Laboratory of Molecular Biology, Cambridge, England.
Harrison
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005609:Free Radicals; D066298:In Vitro Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D005609:Free Radicals; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C075878:ttk protein, Drosophila; C031156:poly d(I-C); D004247:DNA