ROLE OF THE ZINC(II) IONS IN THE STRUCTURE OF THE 3-FINGER DNA-BINDING DOMAIN OF THE SP1 TRANSCRIPTION FACTOR

被引:61
作者
KUWAHARA, J [1 ]
COLEMAN, JE [1 ]
机构
[1] YALE UNIV,DEPT MOLEC BIOPHYS & BIOCHEM,NEW HAVEN,CT 06510
关键词
D O I
10.1021/bi00489a019
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The transcription factor Spl 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 codons 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 Spl containing the C-terminal 165 residues plus 2 from the cloning vector, designated Spl (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 Spl 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)Spl(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. Reaction of only two of the sulfhydryl zinc ligands with the organic mercurial releases 75% of the Zn(II), suggesting that the Zn(II)-induced folding of the three fingers is probably cooperative. Circular dichroism shows the Zn(II)3 protein to contain ~20% α-helix, ~ 20% β-sheet, and ~60% random coil as the secondary structure of the zinc-finger domain. A large part of the secondary structure is lost when the metal ions are removed.© 1990, American Chemical Society. All rights reserved.
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页码:8627 / 8631
页数:5
相关论文
共 20 条
[1]   SYNERGISTIC ACTIVATION BY THE GLUTAMINE-RICH DOMAINS OF HUMAN TRANSCRIPTION FACTOR SP1 [J].
COUREY, AJ ;
HOLTZMAN, DA ;
JACKSON, SP ;
TJIAN, R .
CELL, 1989, 59 (05) :827-836
[2]   ANALYSIS OF SP1 INVIVO REVEALS MULTIPLE TRANSCRIPTIONAL DOMAINS, INCLUDING A NOVEL GLUTAMINE-RICH ACTIVATION MOTIF [J].
COUREY, AJ ;
TJIAN, R .
CELL, 1988, 55 (05) :887-898
[3]   EXAFS STUDY OF THE ZINC-BINDING SITES IN THE PROTEIN TRANSCRIPTION FACTOR-IIIA [J].
DIAKUN, GP ;
FAIRALL, L ;
KLUG, A .
NATURE, 1986, 324 (6098) :698-699
[4]   CONTROL OF EUKARYOTIC MESSENGER-RNA SYNTHESIS BY SEQUENCE-SPECIFIC DNA-BINDING PROTEINS [J].
DYNAN, WS ;
TJIAN, R .
NATURE, 1985, 316 (6031) :774-778
[5]   METAL-DEPENDENT FOLDING OF A SINGLE ZINC FINGER FROM TRANSCRIPTION FACTOR-IIIA [J].
FRANKEL, AD ;
BERG, JM ;
PABO, CO .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (14) :4841-4845
[6]   THE FUNCTION AND STRUCTURE OF THE METAL COORDINATION SITES WITHIN THE GLUCOCORTICOID RECEPTOR DNA-BINDING DOMAIN [J].
FREEDMAN, LP ;
LUISI, BF ;
KORSZUN, ZR ;
BASAVAPPA, R ;
SIGLER, PB ;
YAMAMOTO, KR .
NATURE, 1988, 334 (6182) :543-546
[7]  
FRIED MG, 1981, NUCLEIC ACIDS RES, V13, P6505
[8]   COMPUTED CIRCULAR DICHROISM SPECTRA FOR EVALUATION OF PROTEIN CONFORMATION [J].
GREENFIE.N ;
FASMAN, GD .
BIOCHEMISTRY, 1969, 8 (10) :4108-&
[9]  
HUNT JB, 1984, J BIOL CHEM, V259, P4793
[10]   PROMOTER-SPECIFIC ACTIVATION OF RNA POLYMERASE-II TRANSCRIPTION BY SP1 [J].
KADONAGA, JT ;
JONES, KA ;
TJIAN, R .
TRENDS IN BIOCHEMICAL SCIENCES, 1986, 11 (01) :20-23