ANALYSIS OF INTERACTIONS BETWEEN HUGATA-3 TRANSCRIPTION FACTOR AND 3 GATA REGULATORY ELEMENTS OF HIV-1 LONG TERMINAL REPEAT, BY SURFACE-PLASMON RESONANCE

Relative affinities of transcriptional regulatory elements for their r espective factor have been essentially studied by bandshift analysis. Here we report a realtime study of factor/DNA interactions using a sur face plasmon resonance approach and further characterization of recove red proteins involved in this interaction. For this purpose, human GAT A-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 c hosen, in which only site 2 is a noncanonical GATA site. Direct analys is of sensorgrams, with recombinant huGATA-3, allowed the comparison o f association and dissociation profiles of the three DNA regions and t heir ranking according to their relative affinities. This result, conf irmed by competitions with each GATA site, demonstrated the higher rel ative affinity (at least sevenfold) of site 3. Interactions between th e canonical and unique GATA site 3 and nuclear extracts were also stud ied in real time and provided information on its association and disso ciation rates for native huGATA-3. Finally, recovered protein was iden tified as genuine huGATA-3 by SDS-PAGE, Western blotting, and bandshif t assays. (C) 1997 Academic Press.