Nucleic Acids Research, Vol 25, Issue 5 1050-1055, Copyright © 1997 by Oxford University Press
K Paal, PA Baeuerle and ML Schmitz
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.
ARTICLES
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
Institute of Biochemistry and Molecular Biology, Albert-Ludwigs University, Hermann-Herder Strasse 7, D-79104 Freiburg, Germany.
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