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 Activation
domains are classified according to the preponderance of amino acid residues such as
glutamine, proline, and those bearing acidic side chains. Of these classes, the acidic
activators have been the most extensively studied. Notwithstanding the gains that have
been made in identifying the targets of acidic activation domains and elucidating the
importance of particular residues for their function, the structural basis for the ability
of activation domains to stimulate transcription remains poorly understood. We
demonstrate for the first time that the activation domain of the herpes simplex virus VP16
protein undergoes an induced coil-to-helix transition upon interaction with its target
protein hTAFII31, with residues along one face of the nascent helix making intermolecular
contacts to hTAFII31. |
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To analyze
independently the importance of residues in VP16C for both the interaction with TAF1-140
and activation of transcription, we |
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Each of the
roughly 100,000 genes encoded in the human genome is subject to individual dosage control.
The systems that regulate gene expression respond to a wide variety of developmental and
environmental stimuli, thus allowing each cell type to express a unique and characteristic
subset of its genes, |
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performed in vitro biochemical assays with a series of VP16C deletion mutant proteins (A).
For detection of TAF-binding activity, each deletion mutant was fused to
glutathione-S-transferase (GST) and analyzed for the ability to pull down TAF1-140 (B).
Deletion of residues 475 to 490 from GST-VP16C abolished TAF binding, whereas deletion of
residues 452 to 468 retained binding activity. Further deletion of residues 486 to 490 had
no detectable effect; however, removal of residues 469 to 473 or 481 to 485 resulted in a
loss of binding activity. For assessment of the ability of the deletion mutants to
activate transcription, each mutant was fused to the yeast GAL4 DNA binding domain
(residues 1 to 147) and analyzed for the ability to activate transcription in vitro with
HeLa nuclear extracts (C) (17). GAL4-VP16C stimulated transcription of the reporter
construct containing five GAL4 recognition sites. A fusion construct in which residues 452
to 468 of VP16C were deleted activated transcription as strongly as did GAL4-VP16C itself.
However, deletion of residues 475 to 490 reduced activation potential; this was slightly
above GAL4 activation alone. Although deletion of residues 486 to 490 had no detectable
effect, further deletion of residues 469 to 473 or 481 to 485 reduced transcriptional
activity. Thus, the transcriptional activity of VP16C is directly correlated with the
strength of its binding to TAF1-140; this is consistent with the notion that the
interaction between these two proteins is responsible for the activation signal observed
in our in vitro assays. Moreover, the COOH-terminal segment of VP16C (VP16469-485,
residues 469 to 485) is necessary and sufficient to bind TAF1-140 and activate
transcription in vitro, and it corresponds to the region that was mapped through NMR
experiments to interact with TAF1-140. 
A
long-standing puzzle relates to the specific role of the acidic residues in the acidic
activation domain. Evidence suggests that acidic residues play an important role in the
function of acidic activation domains; however, the results presented here and elsewhere
indicate that hydrophobic residues also play an important role. Although 5 out of
the 17 residues that make up the minimal activation peptide VP16469-485 are acidic, only
one of these, Asp472, exhibits significant perturbation of its -proton chemical shift upon
binding to TAF1-140. Asp472 may make a direct, specific contact; however, this contact is
apparently not conserved in the activation domains of p53 and p65. Moreover, the positions
of the acidic residues in acidic activation domains generally appear to be unimportant.
This seeming paradox can be resolved by a model in which the acidic residues establish
long-range electrostatic interactions with hTAFII31. Such
electrostatic forces would attract basic hTAFII31 over
relatively long distances in solution, thereby increasing the rate at which the activation
domain locates its target. Once the activation domain and hTAFII31
come into close range, the activation domain undergoes an induced structural transition to
an a-helix, thereby enabling the establishment of direct hydrophobic contacts with
nonpolar residues of hTAFII31. Because such folding transitions
are highly cooperative, the coupling of folding to targeting by activation domains
provides a mechanism whereby multiple weak interactions can produce a pronounced biologic
response. 
and to adjust the dosage of
particular gene products as needed. The importance of dosage control is underscored by the
fact that targeted disruption of key regulatory molecules in mice often results in drastic
phenotypic abnormalities, just as inherited or acquired defects in the function of genetic
regulatory mechanisms contribute broadly to human disease. These findings have fueled
efforts aimed at understanding fundamental mechanisms of gene regulation, with a eye
toward discovering means of overriding endogenous regulatory controls or of creating new
signaling circuitry in cells. Of particular interest in this regard are synthetic
molecules designed to modulate gene transcription in living cells. To date, attention has
been focused mostly on the discovery of organic molecules that interact
sequence-specifically with DNA and thereby antagonize transcriptional stimulation by
activator proteins. We have devised a strategy for conditional activation of gene
expression using organic molecules that simultaneously target the transcriptional
machinery and a DNA-binding protein. A molecule designed to serve as an intermediary
between a DNA-binding protein and the transcriptional apparatus should incorporate binding
elements for each of these two macromolecular targets. As the former, we chose the
immunosuppressive drug FK506, which binds with high affinity (Kd = 0.4 nM) to the
immunophilin FKBP12. 
FK506 and certain of its derivatives can be targeted to the DNA
binding domain of GAL4 by fusion of this domain to FKBP (GAL4-FKBP). Modification of the
calcineurin-binding surface of FK506 yields derivatives that lack immunosuppressive
properties but retain the ability to bind FKBP with high affinity. We equipped one such
nonimmunosuppressive FK506 derivative, bearing a hydroxyethyl group at C-21, with an
activator element through the addition of a linker, to which was attached a 29-amino acid
L peptide containing a tandemly repeated undecamer sequence derived from the N-terminal
portion of the VP16 activation domain . 
This
L peptide, when directly fused to the GAL4 DNA-binding domain, is a potent activator of
transcription in vivo, most likely through binding directly to component(s) of the basal
transcriptional apparatus. Thus, the FK506-peptide conjugate L-1 could in principle be
capable of bridging GAL4-FKBP and the basal transcriptional apparatus. We
have shown that a 4 kDa synthetic molecule containing two linked binding elementsone that
targets a DNA-binding protein and another that targets the transcriptional machinerycan
coactivate transcription of a mammalian promoter. Specifically, a designed coactivator
containing a nonnatural completely D-configured peptide stimulates transcription in vitro
with only slightly less potency than the corresponding coactivator bearing the natural L
configuration. Strikingly, the nonnatural molecule D-1 also stimulates transcription of a
GAL4-driven promoter in vivo, when present in conjunction with GAL4-FKBP. These
experiments thus demonstrate both the feasibility of using small molecules to coactivate
gene expression in vitro and in vivo, and the ability of completely nonnatural small
molecules to serve this function.