Analysis of interactions between huGATA-3 transcription factor and three GATA regulatory elements of HIV-1 long terminal repeat, by surface plasmon resonance.
Relative affinities of transcriptional regulatory elements for their respective factor have been essentially studied by bandshift analysis. Here we report a real-time study of factor/DNA interactions using a surface plasmon resonance approach and further characterization of recovered proteins involved in this interaction. For this purpose, human GATA-3, either recombinant or in nuclear extracts, and three natural GATA elements of the HIV-1 long terminal repeat (sites 1, 2, and 3) were chosen, in which only site 2 is a noncanonical GATA site. Direct analysis of sensorgrams, with recombinant huGATA-3, allowed the comparison of association and dissociation profiles of the three DNA regions and their ranking according to their relative affinities. This result, confirmed by competitions with each GATA site, demonstrated the higher relative affinity (at least sevenfold) of site 3. Interactions between the canonical and unique GATA site 3 and nuclear extracts were also studied in real time and provided information on its association and dissociation rates for native huGATA-3. Finally, recovered protein was identified as genuine huGATA-3 by SDS-PAGE, Western blotting, and bandshift assays.
Galio
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CNRS UPR 0415, ICGM, 22, rue Méchain, Paris, 75 014, France.
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Guillet
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Vaquero
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D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D050990:GATA3 Transcription Factor; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D013601:T-Lymphocytes; D015534:Trans-Activators / Q000378:metabolism*; D016335:Zinc Fingers
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators