Optimal Oct-2 affinity for an extended DNA site and the effect of GST fusion on site preference.
The regulator of immunoglobulin expression Oct-2 and the related widely expressed transcription factor Oct-1 have been shown to interact with DNA sequences containing an "octamer" motif, ATGC(A/T)AAT. To better understand Oct-2 function we have used random oligonucleotide selection and competition assays to define the optimal recognition site for this protein. The selected site contains an extended sequence that is remarkably similar to octamer-heptamer sequences found in immunoglobulin heavy-chain regulatory sequences, and the affinity of Oct-2 for this site is at least 50-fold greater than for sites containing the octamer motif alone. Fusion to glutathione S-transferase, a widely used model for protein-DNA and protein-protein interaction, does not alter the optimal Oct-2 recognition site, but inhibits Oct-2 POU-domain dimerization, slows the dissociation rate of the GST-Oct-2/DNA complex, and increases the relative importance of the heptamer domain for Oct-2 binding. These data advance our ability to identify in vivo targets of POU-factor regulation and also suggest that GST-fusion proteins should be used with caution in DNA-binding studies.
Rhee
J M
JM
Department of Psychiatry, University of California, San Diego, La Jolla 92093-0603, USA.
Trieu
M
M
Turner
E E
EE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D050813:Octamer Transcription Factor-2; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D013379:Substrate Specificity; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D015342:DNA Probes; D004268:DNA-Binding Proteins; D050813:Octamer Transcription Factor-2; C058435:POU2F2 protein, human; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA