Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila.
The ventral nervous system defective (vnd)/NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose. Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd/NK-2 affinity column chromatography, cloned, and sequenced. The consensus nucleotide sequence of the vnd/NK-2 homeodomain binding site was shown to be T(T/C)AAGTG(G/C). The apparent equilibrium dissociation constant (K(D)) of the vnd/NK-2 homeodomain for the consensus sequence is 1.9 x 10(-10) M. In addition, results of competition between oligodeoxynucleotides for binding to the vnd/NK-2 homeodomain and determination of the apparent K(D) values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding.
Wang
Lan-Hsiang
LH
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1654, USA.
Chmelik
Rebecca
R
Nirenberg
Marshall
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors
D018076:DNA, Complementary; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D014157:Transcription Factors; C094797:vnd protein, Drosophila; D004247:DNA