LAC9 DNA-binding domain coordinates two zinc atoms per monomer and contacts DNA as a dimer.
The LAC9 protein of Kluyveromyces lactis activates transcription by binding to upstream activating sequences lying in front of genes of the lactose-galactose regulon. LAC9 belongs to a family of fungal proteins having a conserved domain containing 6 cysteines. This domain, termed a C6 zinc finger, is thought to bind one zinc atom and to play a vital role in DNA binding. To further characterize the DNA-binding domain of LAC9, we have developed a procedure to produce and to purify milligram amounts of LAC9 peptides. The two larger peptides, one containing amino acids 1-228 and the other containing amino acids 85-228, formed dimers in solution and bound DNA specifically as a dimer. The smallest LAC9 peptide, amino acids 85-160, failed to dimerize and did not bind DNA. Atomic absorption spectroscopy revealed that each LAC9 monomer coordinated two zinc atoms, not one, as had been predicted. This result suggests, as does previously published data, that the C6 zinc finger domain has a unique conformation that may represent a new type of DNA-binding motif.
Department of Biochemistry, University of Kentucky, Lexington 40536.
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Halvorsen
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Nandabalan
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Dickson
D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D015032:Zinc / Q000032:analysis* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058596:LAC9 protein, Kluyveromyces lactis; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014157:Transcription Factors; D015032:Zinc