BASAL TRANSCRIPTION FACTORS TBP AND TFIIB AND THE VIRAL COACTIVATOR E1A 13S BIND WITH DISTINCT AFFINITIES AND KINETICS TO THE TRANSACTIVATION DOMAIN OF NF-KAPPA-B P65
K. Paal et al., BASAL TRANSCRIPTION FACTORS TBP AND TFIIB AND THE VIRAL COACTIVATOR E1A 13S BIND WITH DISTINCT AFFINITIES AND KINETICS TO THE TRANSACTIVATION DOMAIN OF NF-KAPPA-B P65, Nucleic acids research, 25(5), 1997, pp. 1050-1055
Transactivation domains (TADs) are able to contact several components
of the basal transcription apparatus and co-activator molecules, In or
der to study these interactions in biophysical detail, binding of the
well-characterized TAD from the human transcription factor NF-kappa B
p65 (RelA) to the basal transcription factors TBP and TFIIB and the vi
ral co-activator protein E1A 13S was chosen as a model system to inves
tigate the kinetics and affinities of such protein-protein interaction
s by surface plasmon resonance analysis, The TAD of NF-kappa B p65 sho
wed remarkably different affinities and kinetics in binding to the var
ious proteins, The real-time kinetic measurements revealed an associat
ion rate constant (k(ass)) of 2.3 x 10(6)/M/s for the interaction betw
een 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 prot
ein (4.9 x 10(4)/M/s), The dissociation rate constants (k(diss)) 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 dissoc
iation constants (K-d) differed between 3.4 x 10(-10) M for the strong
ly binding TBP protein and 2.3 x 10(-8) M and 2.6 x 10(-8) M for the w
eaker binding TFIIB and E1A 13S proteins respectively, Non-linear anal
ysis of the appropriate part of the sensorgrams revealed monophasic as
sociation and dissociation kinetics for binding between the p65 TAD an
d all three interaction partners, The remarkable differences in protei
n affinities add another aspect to a more detailed understanding of fo
rmation of the transcription preinitiation complex, The cotransfection
of TBP and E1A 13S stimulated NF-kappa B p65-dependent gene expressio
n, showing the biological significance of these interactions.