Role of the zinc(II) ions in the structure of the three-finger DNA binding domain of the Sp1 transcription factor.
The transcription factor Sp1 from Hela cells contains near the C-terminus of this protein of 778 amino acids three contiguous repeats of an amino acid sequence, -Cys-X4-Cys-X12-His-X3-His-, typical of the Cys2His2-type zinc-finger DNA binding domain first found in transcription factor TFIIIA. A DNA sequence corresponding to the condons from residue 614 to residue 778 of Spl (encompassing the three zinc-finger motifs) has been cloned and overproduced in Escherichia coli. The fragment of Sp1 containing the C-terminal 165 residues plus 2 from the cloning vector, designated Sp1(167*), can be extracted with 5 M urea and then refolded in the presence of Zn(II) to a protein of specific conformation containing 3.0 +/- 0.2 mol of tightly bound Zn(II)/mol of protein. Gel retardation assays using a labeled 14-bp DNA sequence containing a consensus Sp1 binding site show that the refolded Zn(II) protein specifically recognizes the "GC box" sequence in the presence of a large excess of calf thymus DNA. Treatment of Zn(II)Sp1(167*) with 10 mM EDTA results in removal of Zn(II) and the formation of an apoprotein which does not specifically recognize DNA. Cd(II) can be exchanged for Zn(II) in the refolded protein with full retention of specific DNA recognition. This is the first Cys2His2-type "finger" protein where this substitution has been accomplished. Titration of the Zn(II) protein with 6 mol of p-mercuribenzenesulfonate/mol of protein results in the complete release of the three Zn(II) ions. Release of Zn(II) is highly cooperative.(ABSTRACT TRUNCATED AT 250 WORDS)
Kuwahara
J
J
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Coleman
J E
JE
D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015032:Zinc / Q000502:physiology*
D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D002104:Cadmium; D004247:DNA; D015032:Zinc