GATA-1 bends DNA in a site-independent fashion.
The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.
Ghirlando
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Laboratory of Molecular Biology, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892, USA.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D002645:Chickens; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005982:Glutathione Transferase; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D058619:Maltose-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase