Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain.
Transcription factor Sp1 is a protein present in mammalian cells that binds to GC box promoter elements and selectively activates mRNA synthesis from genes that contain functional recognition sites. We have isolated a cDNA that encodes the 696 C-terminal amino acid residues of human Sp1. By expression of truncated fragments of Sp1 in E. coli, we have localized the DNA binding activity to the C-terminal 168 amino acid residues. In this region, Sp1 has three contiguous Zn(II) finger motifs, which are believed to be metalloprotein structures that interact with DNA. We have found that purified Sp1 requires Zn(II) for sequence-specific binding to DNA. Thus, it is likely that Sp1 interacts with DNA by binding of the Zn(II) fingers. To facilitate the identification of mutant variants of Sp1 that are defective in DNA binding, we have also devised a bacterial colony assay for detection of Sp1 binding to DNA.
Kadonaga
J T
JT
Howard Hughes Medical Institute, Department of Biochemistry, University of California, Berkeley 94720.
Carner
K R
KR
Masiarz
F R
FR
Tjian
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000494:pharmacology
D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D015032:Zinc