Basal transcription factors TBP and TFIIB and the viral coactivator E1A 13S bind with distinct affinities and kinetics to the transactivation domain of NF-kappaB p65.
Transactivation domains (TADs) are able to contact several components of the basal transcription apparatus and co-activator molecules. In order to study these interactions in biophysical detail, binding of the well-characterized TAD from the human transcription factor NF-kappaB p65 (RelA) to the basal transcription factors TBP and TFIIB and the viral co-activator protein E1A 13S was chosen as a model system to investigate the kinetics and affinities of such protein-protein interactions by surface plasmon resonance analysis. The TAD of NF-kappaB p65 showed remarkably different affinities and kinetics in binding to the various proteins. The real-time kinetic measurements revealed an association rate constant (kass) of 2.3 x 10(6)/M/s for the interaction between the p65 TAD and TBP. The association rate constants of the p65 TAD were much weaker for TFIIB (6.8 x 10(4)/M/s) and for the E1A 13S protein (4.9 x 10(4)/M/s). The dissociation rate constants (kdiss) were determined to be 7.9 x 10(-4)/s for TBP, 1.6 x 10(-3)/s for TFIIB and 1.3 x 10(-3)/s for the E1A protein. Accordingly, the calculated dissociation constants (Kd) differed between 3.4 x 10(-10)M for the strongly binding TBP protein and 2.3 x 10(-8)M and 2.6 x 10(-8)M for the weaker binding TFIIB and E1A 13S proteins respectively. Non-linear analysis of the appropriate part of the sensorgrams revealed monophasic association and dissociation kinetics for binding between the p65 TAD and all three interaction partners. The remarkable differences in protein affinities add another aspect to a more detailed understanding of formation of the transcription preinitiation complex. The co-transfection of TBP and E1A 13S stimulated NF-kappaB p65-dependent gene expression, showing the biological significance of these interactions.
Paal
K
K
Institute of Biochemistry and Molecular Biology, Albert-Ludwigs University, Hermann-Herder Strasse 7, D-79104 Freiburg, Germany.
Baeuerle
P A
PA
Schmitz
M L
ML
D017500:Adenovirus E1A Proteins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D006801:Humans; D007700:Kinetics; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism*; D015533:Transcriptional Activation
D017500:Adenovirus E1A Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII