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J Biol Chem, Vol. 273, Issue 48, 32080-32087, November 27, 1998
From the Department of Biochemistry, University of Washington,
Seattle, Washington 98195-7350
The yeast transcriptional activator Adr1p
controls expression of the glucose-repressible alcohol dehydrogenase
gene (ADH2), genes involved in glycerol metabolism, and
genes required for peroxisome biogenesis and function. Previous data
suggested that promoter-specific activation domains might contribute to
expression of the different types of
ADR1-dependent genes. By using gene fusions
encoding the Gal4p DNA binding domain and portions of Adr1p, we
identified a single, strong acidic activation domain spanning amino
acids 420-462 of Adr1p. Both acidic and hydrophobic amino acids within
this activation domain were important for its function. The critical
hydrophobic residues are in a motif previously identified in p53 and
related acidic activators. A mini-Adr1 protein consisting of the DNA
binding domain of Adr1p fused to this 42-residue activation domain
carried out all of the known functions of wild-type ADR1.
It conferred stringent glucose repression on the ADH2 locus and on UAS1-containing reporter genes. The putative inhibitory region
of Adr1p encompassing the protein kinase A phosphorylation site at
Ser-230 is thus not essential for glucose repression mediated by
ADR1. Mini-ADR1 allowed efficient derepression
of gene expression. In addition it complemented an
ADR1-null allele for growth on glycerol and oleate media,
indicating efficient activation of genes required for glycerol
metabolism and peroxisome biogenesis. Thus, a single activation
domain can activate all ADR1-dependent promoters.
Activation domains of transcription factors transmit signals to
the transcriptional machinery of a cell to ensure proper gene expression. When tethered to a DNA binding domain, either covalently or
via protein-protein interaction, they ensure that signals are transmitted to appropriate genes to activate their transcription. Although the specificity of gene expression is determined primarily by
the DNA binding domain of transcription factors, activation domains can contribute to this specificity (1-4).
Activation domains function by contacting other proteins that are
components of the transcriptional machinery (5). The proteins contacted
by activation domains include various subunits of TFIID, including
TATA-binding protein itself and TATA-binding protein-associated
factors, and other general transcription factors such as TFIIB, TFIIH,
and members of adaptor or mediator complexes. By contacting these
proteins, they recruit RNA polymerase II to the promoter and facilitate
initiation and elongation of transcription (5-11).
ADR1 encodes a post-translationally regulated transcription
factor in the yeast Saccharomyces cerevisiae that activates
the expression of the glucose-repressed ADH2 gene as well as
genes involved in glycerol metabolism and peroxisome biogenesis
(12-23). As with many eukaryotic transcription factors, Adr1p contains multiple domains. The ADR1 DNA binding domain
(ABD)1 is encompassed by
amino acids ~70-165 (16). A nuclear targeting signal is found in the
first 21 amino acids of the protein (24). Four transcription activation
domains (TADs) were identified by fusion of various parts of
ADR1 to the DNA binding domain of LexA as follows: TADI,
amino acids 1-220; TADII, amino acids 263-359; TADIII, amino acids
359-571; and TADIV amino acids 642-704 (20, 25). In vitro
binding studies, as well as in vivo co-immunoprecipitation, suggest that TFIID, TFIIB, and Gcn5p, a histone acetylase, are targets
of these activation domains (25, 26). In addition, two inhibitory
regions were identified, one of which contains the protein kinase A
phosphorylation site at amino acid Ser-230. Mutation of this site
enhances the activity of Adr1p on both repressing and derepressing
media by an unknown mechanism (27-30).
Deletion studies of ADR1 suggested that its activation
domains were promoter-specific (20, 25). Deletion of the 3'- half of
ADR1, including TADIV, prevented cells from growing on
oleate, an inducer of peroxisome proliferation, whose utilization
requires ADR1 (14). This ADR1 deletion mutant
could still support growth on glycerol, but further deletion of TADIII
prevented growth on glycerol and still allowed ADH2
derepression (14, 20).
Here we describe the results of our search for transcriptional
activation domains in Adr1p by fusing different parts of
ADR1 to the DNA binding domain of GAL4 (GBD). In
contrast to the results obtained using LexA fusions, our analyses
indicate the presence of a single major TAD corresponding to TADIII.
When TADIII was deleted from ADR1, the rate of
ADH2 derepression decreased more than 10-fold without
altering the stability of Adr1p, indicating that it plays an important
role in transcription activation by native Adr1p. We have identified
the region of TADIII that is important for activation as amino acids
420-462. Within this region, the pattern of hydrophobic amino acids
matches that in the transcription activation domains of p53 and related
acidic activators. Mutagenesis showed that hydrophobic amino acids
within the consensus sequence were particularly important for
transcription activation, suggesting a conserved role for this motif in
mammalian and yeast cells.
We examined the function of a gene we call mini-ADR1, a
fusion of TADIII to the DNA binding domain of Adr1p.
Mini-ADR1 activated ADH2 expression and growth on
glycerol or oleate as well as wild-type, full-length Adr1p.
Importantly, mini-ADR1 conferred stringent glucose
repression on the ADH2 gene and on UAS1-containing
reporters. The same amino acids that were important for transcription
activation by GBD-TADIII were important for the activity of
mini-ADR1.
Strains--
Yeast strains are listed in Table
I.
Growth of Yeast Cultures--
Yeast strains were grown in YPD or
synthetic medium (SM) prepared according to standard methods (31).
Glucose-repressed cultures were started in YP or supplemented SM
containing 5% glucose. Derepressing cultures were inoculated into YP
or supplemented SM containing 2% ethanol, 2% glycerol, 1% lactate,
and 0.05% glucose.
Plasmid Construction--
The base plasmid for
GAL4-ADR1 gene fusions was pMA424 (32) an
episomal, HIS3 plasmid containing the ADH1
promoter and terminator, the GAL4 DNA binding domain (amino
acid residues 1-147), and a polylinker containing unique
EcoRI, BamHI, and SalI sites at the 3'
end of the GAL4 fragment. Fragments of ADR1 were
introduced into the polylinker after cutting with EcoRI,
EcoRI and NruI, EcoRI and
BamHI, or EcoRI and SalI. The correct
reading frame at the EcoRI site in the vector and in
ADR1 was obtained using the Klenow fragment of E. coli DNA polymerase or mung bean nuclease, as required. The
junction sequence was verified by DNA sequence analysis using a
GAL4-specific oligonucleotide.
Large fragments of ADR1 containing internal EcoRI
sites were cloned using partial digestion with EcoRI or
serial cloning using unique restriction sites within intermediate
constructs. The source of ADR1 was pADR1-4b,
which contains ADR1 flanked by SalI sites introduced at the NciI and ScaI sites immediately
flanking the 5' and 3' ends of the ORF, respectively, cloned into
pUC19. The ADR1 fragments in plasmid pJSL33 were derived
from E. coli expression vector pCQV229 (16). Other
ADR1 fragments were made by PCR and utilized primers that
generated BamHI and SalI sites at the 5' and 3'
ends of the fragments, respectively. The PCR products were cut with
these enzymes and cloned into pMA424 cut with the same two enzymes.
A deletion of ADR1 representing amino acids 419-467 in
ADR1 was made by recombinant PCR. This deletion was
introduced into a GBD-ADR1 gene fusion representing
ADR1-encoded amino acids 21-753 (deleted for amino acids
419-467) and also into non-fusion ADR1 genes by swapping
DNA fragments, to create plasmids pJSL127 and pACh3. These are
TRP1-CEN-based plasmids based on pBC3T1 (33) and pRS314
(34), respectively, in which ADR1 is expressed from its own promoter.
Mini-ADR1 was constructed in several steps. A 6.2-kilobase
pair fragment containing the ADR1 gene from the
SspI site at position
Mutations were introduced into ADR1-encoded amino acids
420-462 using mutagenic primers and recombinant PCR or the Quick
Change mutagenesis kit obtained from Stratagene. The sequences of the mutagenic primers are available on request. Several mutant clones from
each construction were sequenced. Two clones with the identical mutation were usually tested to reduce the possibility that a cryptic
mutation introduced during the PCR amplification had an effect on the
observed phenotype. The construction of pJS23 (ADR1-VP16 in
pRS314) is described
elsewhere.2
Enzyme Assays--
ADH enzyme activity was visualized after
electrophoresis of protein extracts on non-denaturing polyacrylamide
gels (30). Protein Extracts and Western Blotting--
Protein extracts from
yeast cells were prepared by disrupting the cells with glass beads (400 µm) in 50 mM Tris (pH 6.8), containing 1 mM
phenylmethylsulfonyl fluoride, 2 mg/ml aprotenin, 1 mg/ml leupeptin, 2 mg/ml pepstatin A, and 1% 2-mercaptoethanol at 4 °C. Debris was
removed by centrifugation. A portion of the extract was brought to 10%
glycerol, 1% sodium dodecyl sulfate and heated to 95 °C for 5 min.
Protein concentration in the remaining extract was determined using
Bio-Rad Protein Assay solution (Bio-Rad). Denatured protein (50 µg)
was separated by SDS-polyacrylamide gel electrophoresis. The proteins
were transferred to nitrocellulose. Adr1p was detected with polyclonal
antibodies as previously described (37).
GAL4-ADR1 fusion proteins were detected with
GAL4 GBD (DNA binding domain) monoclonal antibody
(Stratagene, catalog number 5399-1).
DNA Electrophoretic Mobility Shift Assays--
DNA binding and
the electrophoretic mobility shift assays were performed as described
(38).
Identification of a Single ADR1 Activation Domain Using GBD-ADR1
Gene Fusions--
Gene fusions between the GAL4 DNA binding
domain (GBD; amino acids 1-147) and different fragments of
ADR1 were made in the plasmid pMA424 (39). This is a high
copy plasmid containing HIS3 for selection in yeast. The
gene fusion is expressed from the strong ADH1 promoter. A
subset of the plasmids generated is depicted in Fig.
1, which shows the region of Adr1p
present and the activities that were induced by their presence in a
strain containing a GAL7-lacZ reporter gene on a high copy
plasmid (p632-17b-2 (40)).
The first set of gene fusions (Fig. 1, pJSL68 through
pJSL33, and others not shown) suggested the presence of a
strong activation domain between amino acids 336 and 505. Deletion of
the C-terminal 582 amino acids had no significant effect on activation.
More importantly, GBD-ADR1 containing the C-terminal 583 amino acids was inactive (pJSL66). Plasmids encoding the N-terminal 336 or 229 amino acids, both of which contain TADI, were inactive. We tested the effect of mutating Ser-30 to Ala, which leads to
hyper-activation of ADH2 expression. We saw no effect of
this mutation at a UASG
Plasmids pJSL97 through pJSL116 (Fig. 1) were used to more precisely
localize the region responsible for transcriptional activation by
GBD-ADR1 fusions. Their activities indicated that the region responsible for transcriptional activation was contained between amino
acids 420 and 462, the most acidic 40-amino acid stretch within Adr1p
(41). This TAD corresponds to TADIII that was identified by
LexA-ADR1 gene fusions. We deleted this region from a large GBD-ADR1 fusion (Adr1p amino acids 21-753). The activity of
the mutant fusion protein decreased more than 10-fold (pJSL116). The wild-type and deletion mutant proteins were present at comparable levels.3 No activity was detected in strains with pJSL104
(Adr1 amino acids 154-424) or pJSL102 (Adr1p amino acids 468-753).
These plasmids encode GBD-TADII and GBD-TADIV.
Because these plasmids appeared to be unstable in GGY1, we continued
our analysis in strain PJ69-4A (42), which carries a
GAL2-ADE2 reporter gene as well as a GAL7-lacZ
reporter gene. The
To try to achieve more stable expression of the gene fusions, and to
confirm the results shown in Fig. 1, we transferred a selected set of
GBD-ADR1 fusions to a different multicopy plasmid containing
TRP1 as a selectable marker. The set represented all portions of ADR1 except the first 21 amino acids. These
plasmids appeared to be more stable as indicated by an absence of
lethal sectoring. The activities of the gene fusions in the new vector assayed in PJ69-4A confirmed the initial results (Fig.
3). The two transformants containing
fusions that include TADIII (pGBA6 and pGBA8) had the highest
All of the GBD-Adr1p fusion proteins could be detected with anti-GBD
antisera with the exception of the fusion containing amino acids
154-420 of Adr1p. This fusion protein was detected using antisera
directed against the internal portion of Adr1p.3 Although
GBD-Adr1-(154-420) may be less abundant than some of the others, we do
not believe that it contains an important activation domain when fused
to GBD, since a fusion protein containing this region but lacking
TADIII was 20-fold less active than the fusion containing TADIII
(compare pGBA7 with pGBA8 in Fig. 3).
Deletion of TADIII Reduces the Rate and Extent of
Derepression--
If TADIII is an important activation domain at UAS1,
its deletion should reduce derepression significantly at promoters
bearing Adr1p-binding sites. This was first tested using the
GAL4-ADR1 fusions pGBA7 and pGBA8. These gene
fusions should be active at both UASG- and UAS1-containing
promoters since they contain both GBD and ABD. Table
II shows that the UAS1-containing
promoters tested were strongly dependent on GBD-ADR1 and
that deletion of the activation domain reduced activity 5-12-fold.
To test the effect of deleting TADIII in its normal Adr1p context, an
ADR1 gene was constructed lacking this region (pJSL127, ADR1
TADIII was not required for growth on glycerol or oleate. A strain
containing ADR1 Activity and Regulation by a mini-ADR1 Gene--
To assay the
activity of TADIII at ADR1-dependent promoters
in the absence of other potential TADs, a mini-ADR1 gene was
constructed. Mini-ADR1 was expressed from the
ADR1 promoter on a low copy plasmid to ensure normal
expression levels. The activation domain was fused in-frame to the
N-terminal 172 amino acids of Adr1p, which contains the DNA binding
domain and nuclear targeting signal. Mini-ADR1 was tested
for its ability to regulate expression of a UAS1-containing reporter
gene and to regulate ADH2 itself. The reporter gene was
tightly glucose-repressed and was activated to the same high level in
the presence of either wild-type ADR1 or
mini-ADR1 when glucose was removed from the media (Table
III). ADH2 itself was also
tightly repressed and efficiently derepressed by mini-ADR1
as shown by analysis of ADHII activity after separation of the ADH
isozymes by electrophoresis (Fig. 5). In
contrast, ABD itself was completely inactive.
The ability of mini-ADR1 to complement an ADR1
deletion mutant for growth on media containing glycerol or oleic acid
as the sole carbon source was also tested (Fig.
6). Mini-ADR1 could complement both of these defects as well as wild-type, full-length
ADR1. As with activation of ADH2 expression, ABD
alone was inactive in these assays.
Identification of Critical Amino Acid Residues in
TADIII--
TADIII is the major activation domain in Adr1p as defined
by the GAL4-ADR1 fusions. It is a member of the
class of acidic activators based on its high content of acidic residues
(11 acidic versus 1 basic residue) and the absence of amino
acids such as Pro or Gln. Changes in TADIII were made first in
GBD-TADIII, and the activity of the mutants was assessed using a
GAL4-dependent reporter gene. To determine the
importance of charged residues in TADIII for activity, clusters of
mutations were made in three regions (Fig.
7A). The charged residues were
changed to Ala, except for Lys-446 which was inadvertently changed to
Thr in three of the mutants (CR8, CR9, and CR10). Mutating all of the
acidic residues in the N-terminal part of TADIII resulted in a 2-fold
reduction in activity (CR1), whereas a 4-fold reduction in activity
resulted when four acidic residues were changed to Ala in the middle of TADIII (CR4). The most severe reduction in activity, about 10-fold, resulted when all five charged residues were mutated at the C terminus
of TADIII (CR10). It appears that negative charge at the C terminus of
TADIII is most important for activation, but the other clusters of
acidic residues also contribute modestly to activity.
The most important hydrophobic amino acids appeared to be in the N
terminus of TADIII (Fig. 7B). Changing pairs of hydrophobic residues in this region reduced activity 20-fold (HA1 and HA2). The
effect of mutating hydrophobic residues in the rest of TADIII was not
as deleterious, although all of the mutations reduced activity significantly.
To test the effect of activation domain mutations in their normal
context, and at different UAS1-containing promoters, hydrophobic residues and the cluster of charged amino acids at the C terminus of
TADIII were substituted by other amino acids, usually Ala, in
mini-ADR1 and full-length ADR1. The effect of the
mutations on a UAS1-containing reporter gene (Table
IV) showed that hydrophobic residues
Phe-425 and Leu-428 and Leu-429 are particularly important. The cluster
of charged amino acids between Lys-446 and Asp-454 was essential for
activity of mini-ADR1, as it was for GBD-TADIII.
The mini-ADR1 mutants were also assessed for their ability
to grow on glycerol or oleate as a carbon source (summarized in Table
IV). The mutations had an effect on growth that was similar to their
effect on activation of the reporter gene. Mini-ADR1 containing substitutions in Phe-425, and Leu-428, Leu-429, or the
cluster of charged resides between Lys-446 and Asp-454 was unable to
complement an ADR1-null allele for growth on glycerol- or
oleate-containing media. Thus, the same residues are probably important
at all UAS1-containing promoters. A qualitatively similar effect of
these mutations was seen at the ADH2 promoter as assessed by
separation of the ADH isozymes by electrophoresis.3
The level of mini-ADR1 mutant proteins in yeast was assessed
by Western blotting. All of the proteins could be detected and were
present at comparable levels, except the two with the mutations CR9 and
CR10, which were present at lower levels than the others.3
To demonstrate that the mutant proteins were properly folded, DNA
electrophoretic mobility shift assays were performed. As shown in Fig.
8, all of the defective mutant proteins
were able to bind DNA. The mutant proteins containing multiple changes
of charged residues (CR9 and CR10) were present at reduced levels as
judged by this assay. However, the active mutant (CR9) was reduced to the same extent as the inactive mutant (CR10), suggesting that the loss
of activity was not due to lower abundance.
The activity of full-length Adr1p containing many of these same
mutations was determined by activation of a reporter gene (Table IV).
Its activity was significantly reduced by several of these mutations,
but the impact of the mutations was not as strong as in the context of
mini-ADR1.
GAL4(GBD)-ADR1 fusions identified a single,
strong activation domain comprised of only 42 amino acids (residues
420-462) within Adr1p. A previous analysis using
LexA-ADR1 gene fusions identified four activation
domains including one between residues 359 and 571. The latter region
was designated TADIII (20). Based on our unpublished
results,3 a later report (25) tested residues 420-462 and
showed that they were sufficient to activate transcription when fused
to LexA. TADIII can function at three natural
GAL4-dependent promoters, at artificial
LexA- and GAL4-dependent promoters,
and at ADR1-dependent promoters. Surprisingly,
TADs I, II, and IV did not significantly activate transcription from
GAL4-dependent promoters.
TADIII is sufficient when fused to the nuclear targeting and DNA
binding domains of ADR1 to carry out all of the known
functions of ADR1. The observation that mini-ADR1
could fully complement an ADR1-null allele was unexpected
since the encoded protein is only 24 kDa, much smaller than full-length
Adr1p (150 kDa). Recently we have found that when TADIII is replaced by
the activation domain from the herpesvirus VP16 gene the
resultant ADR1-VP16 fusion behaved in an identical manner to
mini-ADR1.2
These results argue strongly that regulation mediated by
ADR1 is due to the DNA binding domain of the protein or its
binding site, UAS1 (40). Previous studies of Adr1p have failed to find a regulatory domain that mediates glucose repression. In particular, attempts to demonstrate a regulatory role for phosphorylation at the
cAPK site at amino acid 230 have not met with success (20, 27, 29, 30,
37). Our studies with mini-ADR1 and with
ADR1-VP162 demonstrate that neither the Ser-230
region nor the other portion of Adr1p that functioned as an inhibitory
domain in Lex-ADR1 gene fusion (20) are essential for
glucose repression mediated by Adr1p.
The observation that mini-ADR1 or an ADR1
(ABD)-VP16 gene fusion can complement an
ADR1-null allele also demonstrates that all
ADR1-dependent promoters can be activated by a
single, small activation domain. Thus, it seems unlikely that wild-type
Adr1p employs promoter-specific activation domains.
Also surprising was the high level of activity shown by TADIII even
when expressed from the weak ADR1 promoter on a low
copy plasmid. TADIII was as strong as the activation domain of VP16. This provided us with a sensitive assay to measure the effect of
mutations within the activation domain on the activation of UAS1-containing reporters and on ADH2 itself.
Mutational analysis of the activation domain allowed us to identify a
potential target of TADIII. Several activation domains from
multicellular eukaryotes possess a conserved motif,
FXX Co-crystal structures of the activation domains of VP16 and p53
complexed with hTaf31 and Mdm2, respectively, have demonstrated a role
for the motif FXX TADs have numerous targets in the nucleus (5, 6, 9, 10, 25, 46-48).
One potential target is a histone acetylase complex. In
vitro expressed GST-GCN5 and/or TFIIB interact with ADR1 TADs, and ADR1-dependent gene
expression exhibited ADA2 and GCN5 dependence
(25). Adr1p also interacts with TFIID in vivo as shown by
co-immunoprecipitation, and specific components of TFIID that contact
TADI (TAFIIs 25 and 90), TADII (TATA-binding protein,
TAFII130/145), and/or TADIV (TAFIIs 25, 60, 90, 130/145 and TATA-binding protein) were identified in the same study
(26). No target for TADIII was found.
The fact that ADR1 encoded-TADs interact with multiple
components presumably allows a higher level of transcription activation than would be possible if it interacted with a single factor. Therefore, it is surprising that mini-ADR1, containing only
a single activation domain, was as active as the entire protein. However, TADIII cannot be the only TAD active at
ADR1-dependent promoters since its deletion from
the entire protein reduced (about 10-fold) but did not abolish
expression from these promoters. We speculate that in the context of
the entire protein some of the ADR1-encoded TADs may be
unavailable initially for interaction with components of the
transcription apparatus and that during assembly of an active
initiation complex (49), or during DNA binding (50), different TADs may
become accessible to contribute to activation. By exposing TADs as
artificial proteins, such as in mini-ADR1, they may exhibit
higher activities than they possess in their natural environment. This
may explain why mutations that severely impair the activity of
mini-ADR1 are less deleterious in the context of the native protein.
Mutational analysis of TADIII identified important acidic and
hydrophobic residues (Fig. 9). Changing hydrophobic amino acids to Ala
had a more detrimental effect on activation than individual acidic-to-Ala substitutions, as has been observed for other acidic activators (46, 51-56). Mutation of a cluster of charged (four acidic
and one basic) residues at the C terminus of TADIII inactivated it.
Mutating other clusters of acidic residues also significantly reduced
transcription activation. Based on biochemical studies, it has been
proposed that negatively charged regions of an activation domain might
provide electrostatic interactions between an activation domain and its
target, allowing weak initial binding that is supplanted by an induced
fit interaction between hydrophobic residues on the two partners (43).
There appears to be a correlation between negative charge of TADIII and
transcriptional activity, but further mutagenesis and analysis is
needed to test this rigorously. No promoter-specific defects were noted
for any of the mutants tested. They had similar defects when tested in
the context of a GBD-TADIII fusion or in mini-ADR1 and at
different GAL4- and ADR1-dependent promoters. Thus, it is likely that they are defective in steps common
to all of the promoters tested.
Why was only one ADR1-encoded TAD evident when assayed in
the GAL4 gene fusions whereas four were found using
LexA-ADR1 fusions? Several explanations are possible.
(a) TADI, TADII, and TADIV might be promoter-specific,
activating Lex operator-containing reporters but not
UASG-containing reporters(4). (b) LexA-Adr1p fusions might be more abundant than GBD-Adr1p fusions. As shown here,
ABD itself is inactive when expressed at physiological levels. However,
when expressed at very high levels, its activity can be detected in
derepressed conditions (16) or in strains with mutations in
ADR7, ADR8, or ADR9 (57). Thus, TADI
seems to require very high levels in order to display its activity.
(c) TADI, TADII, and TADIV might be cryptic activation
domains whose activity is strongly dependent on the particular fragment
of ADR1 that is fused to the DNA-binding fragment. This is
true for GAL4-ADI (39) and for ADR1 TADIV (25).
(d) The GAL4-ADR1 fusions encompassing TADI, TADII, and TADIV might have been inactive because they were misfolded. This seems unlikely because deletion of TADIII reduced 10-20-fold the activity of a GBD-ADR1 gene fusion that
would still encode TADs I, II, and IV and yet the mutant protein was as
stable as the wild-type fusion protein.
The interpretation we favor to explain these different results invokes
the ability of activation domains to function with different
efficiencies at different core promoters (4). We suggest that the LexA
promoter used in the other studies recruits a different constellation
of RNA polymerase II-associated factors than the GAL4- and
ADR1-specific promoters we used or that the relative
importance of the various factors differs at different core promoters
(4). Since Adr1p deleted for TADIII can still activate transcription,
albeit weakly, contacts mediated by other TADs are sufficient for
activated transcription.
Why does ADR1 encode 1323 amino acids if its essential
functions can be carried out by mini-ADR1, encoding only 210 amino acids? A similar conundrum is presented by Gal4p, another large transcription factor (881 amino acids) whose essential features are
present in a mini-GAL4 comprised of the DNA binding and
activation/Gal80p-association regions (39, 58). Dissection of
GCN4 also revealed redundant activation domains, only one of
which was necessary and sufficient for nearly wild-type function (52).
In a natural setting the other parts of these transcription factors may
be important. Evolution may have favored the formation of large
transcription factors so that they could perform different or
overlapping functions at different promoters. Our laboratory-based
experiments may only survey a small fraction of these functions. Global
surveys of Adr1p function should allow us to test this hypothesis using
the different ADR1 alleles described here.
*
This work was supported by Research Grant GM 26079 from the
NIGMS, National Institutes of Health (to E. T. Y.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
ABD, ADR1-DNA binding domain (amino acids 70-160); GBD, GAL4-DNA binding domain (amino acids 1-147); PCR, polymerase chain reaction; ORF, open reading frame; TAD, transcription
activation domains; UAS, upstream activation sequence.
2
J. Sloan and E. T. Young, manuscript
submitted for publication.
3
E. T. Young, J. Saario, N. Kacherovsky, A. Chao, J. S. Sloan, and K. M. Dombek, unpublished observations.
Characterization of a p53-related Activation Domain in Adr1p That
Is Sufficient for ADR1-dependent Gene
Expression*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Yeast strains
1067 (from the start of translation)
to the PstI site at position 5226 was assembled between the
EcoRV and PstI sites in pRS314 to create pJS20.
The ADR1 gene in pJS20 was truncated at amino acid 172 by
digesting the plasmid with EcoRI and BamHI and
inserting a 482-base pair EcoRI-BamHI fragment
from pADR1-(172)-lacZ (16) to create pJS21. The truncated
ADR1 gene in pJS21 was fused to ADR1-encoded
amino acids 420-462 to create pJS22. This was done by cutting pJS21
with XbaI, blunt-ending the termini with the Klenow fragment
of DNA polymerase, cutting with BamHI, and recombining the
larger DNA fragment with a BamHI-EcoRV fragment
cut from plasmid pJSL105 (Fig. 1). This plasmid was then cut with
BamHI and the termini blunt-ended and religated. The
BamHI site in pJSL105 is in the pMA424 polylinker, and the
EcoRV site is in the ADH1 terminator. There are 9 and 14 non-ADR1-encoded amino acids between amino acids 172 and 420 and at the C terminus, respectively, that are due to the
polylinkers used for cloning.
-Galactosidase activity was measured either in
permeabilized cells or in cell extracts (36). Generally three
transformants were assayed for each plasmid construct, and the
experiment was repeated at least twice. All of the
GAL4-ADR1 fusions were assayed in at least two
different strains that carried different GAL reporter genes. Although
the absolute values of the activity differed significantly in the
different strains, the relative activity was similar in all strains. In
particular, none of the GAL4-ADR1 fusions was inactive in one strain and active in another.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
GAL4-ADR1 fusions identify a
single activation domain in Adr1p. ADR1 is shown
schematically at the top of the figure. Restriction enzyme
sites used to generate the plasmids are shown above the
representation of ADR1 (E, EcoRI;
S, SphI; N, NruI;
B, BamHI). The numbers
below the indicated site refer to the amino acid position
within the 1323-amino acid ORF. AD, activation domain. The
blue box refers to the GAL4 DNA binding domain
(amino acids 1-147). The plasmids are derived from pMA424. The gene
fusions are expressed from the ADH1 promoter and are
followed by the ADH1 terminator. Plasmids pJSL68 through
pJSL33 were made using restriction enzyme sites; plasmids pJSL97
through pJSL116 were made using using PCR amplification as described
under "Experimental Procedures." The numbers following
the plasmid names correspond to the ADR1-encoded amino acids
fused to GBD. The plasmid pJSL116 contains an in-frame deletion of
amino acids 419-467. The host strain was JSY55 containing the
multicopy plasmid 632-17b-2 which contains two copies of
GAL4 site 3 upstream of a UAS-less GAL1 promoter
fused to lacZ. Similar activities were obtained in a related
strain (GGY1::SV15) containing an integrated reporter
containing a single UASG consensus site upstream of a
CYC1/lacZ fusion. -Galactosidase activities are in Miller
units, and the average deviation was about 30%.
containing reporter in three
different GBD-ADR1 gene
fusions.3
-galactosidase activities in the transformants
were similar to those shown in Fig. 1.3 Only those
transformants that were -galactosidase+ were also
Ade+, as expected (Fig.
2A). However, the
Ade+ colonies showed strong sectoring, both of the Ade
phenotype and for viability (Fig. 2B), indicating that the
active fusion protein is deleterious to the cell. Western blotting of
extracts of transformants carrying these plasmids showed low levels of
some of the fusion proteins3 that could be due to this
toxic effect.
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Fig. 2.
TADIII causes plasmid instability. The
phenotype of plasmids containing GBD-ADR1 was assessed in
strain PJ69-4A (Table I) which contains GAL4-responsive
reporter genes driving expression of lacZ, ADE2,
and HIS3. The ADR1 amino acids encoded in the
gene fusion is listed next to the sector containing a transformant with
the respective plasmid. Most of the plasmids tested are shown in Fig.
1. A, ade , synthetic drop-out plate
lacking adenine; his, synthetic drop-out plate
lacking histidine. The his plate is a control showing
that all of the transformants grew equally well under his
selection alone. B, magnified images of portions of a YPD
plate on which transformants containing the plasmids encoding the
indicated portions of Adr1p were streaked.
-galactosidase activities. Activation of gene expression could also
be scored by growth phenotypes in PJ69-4A since it has
GAL2-ADE2 and GAL1-HIS3 reporter genes. In addition, the Ade phenotype could be scored by color. As expected the
-galactosidase+ transformants were white and the
-galactosidase transformants were red, indicating that
they were Ade+ and Ade, respectively. The
-galactosidase+, Ade+ transformants were
also His+, indicating that TADIII was able to activate
three different GAL4-dependent promoters. The transformant
containing pGBA7, which encodes amino acids 21-753 with an internal
deletion of TADIII, was weakly Ade+ and His+.
However, -galactosidase activity in these transformants was about
20-fold lower than that in the transformants containing pGBA8. Since
the -galactosidase activities in all the other transformants were
even lower, we conclude that TADI, TADII, and TADIV are less than 5%
as active as GBD fusions as TADIII.
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Fig. 3.
An ADR1 activation domain is
necessary and sufficient for activation from multiple GAL
promoters. ADR1 is represented as in Fig. 1.
GAL4-ADR1 fragments are present in a derivative
of pRS304 to which a 2-µm origin of replication has been added (gift
of G. Zhu). The host strain was PJ69-4A. -Galactosidase activities
are in enzyme units (micromoles of ONPG hydrolyzed per min per mg of
protein). The average deviation was about 30%. Cultures were grown to
mid-log phase in trp synthetic medium containing 5%
glucose. Protein extracts were prepared as described under
"Experimental Procedures." Ade and His phenotypes were scored on
trpade and
trphis drop-out plates. The color of the
colonies on trp plates is recorded in
parentheses in the Ade phenotype column: R, red
(Ade); W, white (Ade+). The
his plate contained 10 mM 3-aminotriazole to
increase the stringency of the test.
TADIII is important at UAS1-containing reporters
TADIII: GBD-ADR1(21-753TADIII), and pMA424 (, GBD
alone). Three transformants were grown in derepressing medium that was
selective for both both reporter and activator plasmids as described
under "Experimental Procedures." -Galactosidase activity is
given in Miller units. The values are the averages from three
transformants, and the range of values was ±30%. ADR1 and
TADIII dependence is calculated as the ratio of -galactosidase
activities ± ADR1 and ±TADIII, respectively.
TADIII). The mutant gene was present as a low copy
plasmid and expressed from the native ADR1 promoter. The
rate of derepression of a UAS1-driven lacZ reporter gene was
reduced about 15-fold in the presence of ADR1TADIII
compared with wild-type ADR1. The final level of activation
was 5-fold lower than the level in the presence of wild-type Adr1p
(Fig. 4). Western blot analysis of the
wild-type and mutant proteins indicated that they were present at
comparable levels and at the same level as Adr1p expressed from its
normal chromosomal locus (inset to Fig. 4).
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Fig. 4.
TADIII enhances the rate and extent of
derepression of a UAS1-containing reporter gene. Cultures of
strain TYY303 (adr1 ) containing pKD16 (ADR1), pJSL127
(ADR1TADIII), or pKD8 (vector) and pHDY10, a
UAS1-containing lacZ reporter gene (Table III) were grown in
ura trp synthetic medium containing 5%
glucose. Cells were collected by centrifugation and resuspended in
duplicate cultures of synthetic medium containing derepressing carbon
sources at time 0. At the indicated times samples were withdrawn and
assayed for -galactosidase activity in Miller units. The values
shown indicate the average enzyme activities from samples taken from
the duplicate cultures. Squares, wild-type ADR1;
triangles, ADR1 TADIII; circles,
vector alone. The inset shows a Western blot of Adr1p after
12 h derepression. Extracts were made from strain TYY303
containing pKD8 (vector, lane 1), pKD16 (ADR1,
lane 2), pJSL127 (ADR1TADIII, lane
3), or from strain W303-1A which contains wild-type
ADR1 at its chromosomal locus (lane 4).
TADIII could grow on media containing
these carbon sources,3 indicating that other portions of
ADR1 could compensate for loss of TADIII.
Mini-ADR1 confers stringent glucose repression on a UAS1-containing
reporter gene
1):pHDY10
(CYC1(UAS1)/lacZ) was transformed with the
plasmids pJS20 (ADR1), pJS22 (mini-ADR1), and
pJS21 (ABD; ADR1-encoded amino acids 1-172). The strains
were grown in repressing (r) and derepressing (dr) media selective for
both plasmids (trpura) as described under
"Experimental Procedures." -Galactosidase activities are in
Miller units. The values are the averages from three transformants and
the range of values was ±30%.
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Fig. 5.
Mini-ADR1 stringently regulates
ADH2 expression. Cultures of strain TYY303
(adr1 1) containing pJS22 (mini-ADR1), pJS20
(ADR1), pJS21 (ABD), or pRS314 (vector) were grown in
repressed and derepressed conditions, and extracts were prepared and
analyzed by polyacrylamide gel electrophoresis and staining for ADH
enzyme activity. The pattern shows the ADH isoenzymes after separation
by electrophoresis and staining for enzyme activity in situ.
ADHI, alcohol dehydrogenase I, the constitutive,
ADR1-independent isoenzyme; ADHII,
ADR1-dependent isoenzyme II.
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Fig. 6.
Mini-ADR1 complements an
ADR1-null allele for growth on glycerol or oleate.
Cultures of strain TYY303 (adr1 1) containing pJS22
(mini-ADR1), pJS20 (ADR1), pJS21 (ABD), or pRS314
(vector) were grown in derepressed conditions and then streaked on
trp drop-out plates containing glucose, glycerol, or
oleate as the sole carbon source.
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Fig. 7.
Alanine mutagenesis of TADIII. Changed
residues are shaded. Strain PJ69-4A was transformed with
plasmids carrying GBD-ADR1 gene fusions with the mutations
shown. The base plasmid for mutagenesis was pGBA6 (Fig. 3).
-Galactosidase activity was measured in Miller units and is shown as
a percentage of the activity measured for the wild-type sequence. The
average deviation was about 30%. A, mutation of charged
residues. Numbers 1-3 above the amino acid sequences
indicate the regions where clusters of charged amino acids, Glu or Asp
except in the case of Lys-446, were mutated. Dots
above the sequences indicate the charged residues.
B, mutation of bulky hydrophobic residues. Dots
above the sequences indicate the phenylalanine and leucine
residues that were changed.
Mutations in TADIII alter ADR1-dependent phenotypes
) transformed with the reporter plasmid pHDY10
(CYC1(UAS1)/lacZ). Mutations HA2, HA3, HA5, CR9,
and CR10 are described in Fig. 7. The triple HA mutant is
F425A,L428A,L429A. Transformants were grown selectively
(trpura derepressing medium) as described under
"Experimental Procedures." -Galactosidase activities (Miller
units) of three transformants are reported as the percentage of the
value for WT. The growth phenotype was tested for TYY303 transformed
with pJS22 (mini-ADR1) and mutant derivatives of miniADR1 on
SM (trp) plates containing either glycerol (at 37 °C, 2 days) or oleate (at 25 °C, 3 days). Growth was scored as follows:
+++, growth equivalent to WT; ++ and +, intermediate growth; +/,
hardly detectable growth; , no growth. N C, no construct.
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Fig. 8.
Inactive TADIII mutant proteins are stable
and able to bind DNA. Yeast extracts were prepared from cultures
of strain TYY69 (adr1 ) containing pJS22 (mini-ADR1)
plasmids with mutations in the activation domain. The DNA probe was an
oligonucleotide containing two Adr1p-binding sites (UAS1) to which
Adr1p binds as two monomers. CI, mini-Adr1p single complex;
CII, mini-Adr1p, dimer complex; F, free probe;
NS, nonspecific complex.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, where X is any amino acid and is a
hydrophobic amino acid (43). This motif is also present in TADIII, as
shown in Fig. 9. Related sequences,
FXLLX and GXXLLX, are also
present. Most importantly, the conserved hydrophobic amino acids have
been shown by mutational analysis to be important for the function of
the activation domain. The conservation of this motif and its
importance in transcriptional activation domains from mammals and yeast
suggest that its function may be conserved as well.
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Fig. 9.
A conserved activation domain motif is
present in TADIII. The gene name, position of the motif, and its
sequence are shown. At the bottom of the figure the amino
acid sequence of TADIII and its position in the ADR1 ORF are
shown with the conserved motifs overlined.
Circles below the sequence indicate hydrophobic
residues shown by mutagenesis to be important for function.
Brackets indicate clusters of charged, mostly acidic,
residues whose mutation significantly reduced transcriptional
activation. Residues indicated by closed symbols were more
important for function than residues indicated by open
symbols.
in interaction of an induced 
helix of the activation domain with its partner (43, 44). These results
suggest that this motif in ADR1 might interact with a protein related to either Mdm2 or Taf31. The yeast homolog of TAF31 is TAF17, an essential yeast gene (45). We are
currently investigating the possibility that TADIII
interacts TAF17.
FOOTNOTES
To whom correspondence should be addressed: University of
Washington, Dept. of Biochemistry, Box 357350, Seattle, WA 98195-7350. Tel.: 206-543-6517; Fax: 206-685-9144; E-mail:
ety{at}u.washington.edu.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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