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Molecular and Cellular Biology, September 1998, p. 4971-4976, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Yeast Coactivator MBF1 Mediates GCN4-Dependent
Transcriptional Activation
Ken-ichi
Takemaru,1
Satoshi
Harashima,2
Hitoshi
Ueda,1 and
Susumu
Hirose1,*
Department of Developmental Genetics, National Institute of
Genetics, and Department of Genetics, The Graduate University for
Advanced Studies, Mishima, Shizuoka-ken
411-8540,1 and
Department of
Biotechnology, Graduate School of Engineering, Osaka University,
Suita, Osaka 565,2 Japan
Received 2 April 1998/Returned for modification 18 May
1998/Accepted 29 May 1998
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ABSTRACT |
Transcriptional coactivators play a crucial role in gene expression
by communicating between regulatory factors and the basal transcription
machinery. The coactivator multiprotein bridging factor 1 (MBF1) was
originally identified as a bridging molecule that connects the
Drosophila nuclear receptor FTZ-F1 and TATA-binding protein
(TBP). The MBF1 sequence is highly conserved across species from
Saccharomyces cerevisiae to human. Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4
and TBP. These findings indicate that the coactivator MBF1 functions by
recruiting TBP to promoters where DNA-binding regulators are bound.
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INTRODUCTION |
Studies on transcriptional control
in eukaryotes have revealed many regulatory proteins that bind to
control elements on DNA (17, 25). Typical regulators such as
GAL4 and GCN4 consist of two domains (29), a DNA-binding
domain that binds to a control element in a sequence-specific fashion
and a transcriptional activation domain that somehow stimulates the
basal transcription machinery. A variety of observations have led to
the proposal that some transactivation domains may facilitate binding
of TATA-binding protein (TBP) to a promoter (32, 37
see reference 35 for a review). In many cases, a
member of another class of transcription factors, termed coactivators,
adapters, or mediators, is necessary to connect a regulatory protein
and a component of the basal transcription machinery (see references
23 and 30 for reviews). Recent
studies demonstrated the importance of these non-DNA-binding
transcription factors in gene expression (1, 4, 5, 11, 14, 19, 20,
27, 31).
In in vitro transcription studies, it was found that an insect
coactivator, multiprotein bridging factor 1 (MBF1), can recruit TBP to
a promoter carrying the FTZ-F1-binding site by interconnecting FTZ-F1
and TBP (24, 34). A homology search of the databases showed
that the MBF1 sequence is highly conserved across species from
Saccharomyces cerevisiae to human (34). A key
question concerning MBF1, and its close relatives, is its in vivo
function. To address this issue, we have started genetic studies of
MBF1. We report here that MBF1 serves as a coactivator of GCN4 in the yeast S. cerevisiae.
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MATERIALS AND METHODS |
Yeast strains and plasmids.
The GCN4 disruptant
used in this study was KY803 (trp1-
1 ura3-52 leu2-P1
gcn4-1) (13). Wild-type strain KT130
(trp1-1 ura3-52 leu2-P1) was constructed by homologous
recombination after introduction of GCN4 genomic DNA into
KY803. KT130 was used to generate the mbf1 strain by
replacement of the sequence encoding amino acid residues 64 to 131 of
yeast MBF1 (yMBF1) (34) with a LEU2 selectable
marker.
To construct pMBF1, the 3-kb EcoRI genomic
fragment encompassing the entire MBF1 regulatory and coding
regions was subcloned into YCplac33 (9). YCp88
(13), a centromeric vector containing the constitutive
DED1 promoter, was used to express GCN4 or its mutants
in KY803. pYCGCN4, pYCGCN4bZIP, and
pYCGCN4bZIP carry the entire coding region of GCN4, the
coding region excluding bZIP (amino acid positions 1 to 220), and the
coding region including bZIP (amino acid positions 221 to 281),
respectively. pYCGCN4AD lacks the coding region of the
major activation domain (amino acid positions 88 to 147) of GCN4. To
express yMBF1, its point mutant carrying D112A, and yMBF1NT in the
mbf1 mutant, each coding region tagged with the
GAPDH promoter was inserted into YCplac22 (9) to
yield pYCMBF1, pD112A, and pMBF1NT.
For overexpression of yMBF1 and the D112A mutant under the control of
GAPDH promoter, each coding region tagged with the
GAPDH promoter was cloned into YEplac112 (9) to
yield pYEMBF1 and pYED112A. yTBP was
overexpressed from pyTBP under the control of the
ADH promoter in pDB20 (3). Point mutations
of GCN4 and MBF1 were introduced by site-directed PCR mutagenesis.
Expression of proteins in bacteria.
To produce yMBF1, GCN4,
or its mutant derivatives in Escherichia coli, each
NdeI-BamHI tagged open reading frame was
subcloned into 6HisT-pET11d (Novagen). GCN4bZIP and GCN4bZIP consist
of amino acid positions 1 to 221 and 222 to 281 of GCN4, respectively. For GCN4 and its derivatives, the sequence encoding hemagglutinin (HA)
epitope (8) was inserted in frame at the NdeI
site to generate HA epitope-tagged protein. Histidine-tagged yTBP was expressed as described previously (12). These
histidine-tagged proteins were recovered in a soluble fraction and
purified with Ni-nitrilotriacetic acid resin (Novagen) followed by Mono
S (Pharmacia) chromatography. To produce glutathione
S-transferase-yMBF1 (GST-yMBF1) and its mutant derivatives
in E. coli, each BclI tagged coding region was
subcloned into the BamHI site of pGEX-2T (Pharmacia). GST-yMBF1NT and GST-yMBF141-119 lack the N-terminal 40 amino acids and the amino acid residues from 41 to 119, respectively, of
yMBF1. GST and GST fusion proteins were purified by
glutathione-Sepharose 4B (Pharmacia) chromatography.
GST pull-down assays.
GST or GST fusion proteins (200 ng)
were incubated with bacterially expressed and purified target protein
(100 ng) at 4°C for 1 h in 20 µl of protein-binding buffer
(PBB) (20 mM HEPES-KOH [pH 7.9], 20% glycerol, 0.5 mM EDTA, 60 mM
KCl, 6 mM MgCl2, 0.1% Nonidet P-40, 5 mM
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride). After
incubation, 1 µl (packed volume) of glutathione-Sepharose 4B
(Pharmacia) in 20 µl of PBB was added, and the mixture was rolled at
4°C for 1 h. The beads were collected and washed twice, with 1.5 ml of PBB with rotation at 4°C for 20 min each time, and then bound
proteins were eluted for sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). HA epitope-tagged GCN4 and its derivatives
and yTBP were detected on immunoblots by an anti-HA antibody
(Boehringer GmbH, Mannheim, Germany) at 5 µg/ml and an anti-yTBP
antibody (Upstate Biotechnology) at a 1:50,000 dilution, respectively.
To examine the recovery of the GST and GST fusion proteins, all
pull-down blots were probed with an antibody against GST. More than
95% of the input proteins were recovered.
Electrophoresis mobility shift assays.
DNA-binding assays
for GCN4 and its derivatives were carried out in reaction mixtures (10 µl) containing 12 mM HEPES-KOH (pH 7.9), 5 mM MgCl2, 70 mM KCl, 1 mM dithiothreitol, 6% glycerol, 1 mg of poly(dI-dC) · poly(dI-dC) (Pharmacia) per ml, 5 mg of bovine serum albumin (BSA) per
ml, 1 ng of bacterially expressed GCN4 or its derivative, and 10 fmol
of the probe. The samples were incubated at 30°C for 30 min and then
electrophoresed at room temperature and 100 V for 2.5 h on a 0.8%
agarose gel in Tris-borate-EDTA (TBE). The sequence of the GCN4-binding
site probe was 5'-TCCACCTAGCGGATGACTCTTTTTTTTTCTTAG-3'. The
binding of yTBP to the TATA element was carried out in reaction
mixtures (10 µl) containing 12 mM HEPES-KOH (pH 7.9), 4 mM
MgCl2, 60 mM KCl, 12% glycerol, 2% polyvinyl alcohol
(average molecular weight, 10,000; Sigma), 0.5 mg of poly(dG-dC)
· poly(dG-dC) (Pharmacia) per ml, 5 mg of BSA per ml, 10 ng of
bacterially expressed yTBP, and 5 fmol of the probe. After incubation
at 30°C for 30 min, the samples were electrophoresed at 22°C and
100 V for 2.5 h on a 1% agarose gel in 0.5× TBE containing 2 mM
MgCl2. The sequence of the TATA element probe was
5'-GAATTATACATTATATAAAGTAATGTGATTTC-3'.
S1 nuclease analyses.
Total RNA was isolated by a hot-phenol
method (22) from yeast cells grown in synthetic complete
medium lacking histidine and then treated with 30 mM aminotriazole (AT)
for 6 h. Portions of the RNA (40 µg) were subjected to S1
nuclease analyses with 32P-labeled oligonucleotide probes
for HIS3 and DED1 genes as described previously
(15).
Western analyses.
Yeast protein extracts were prepared from
the histidine-starved cells grown as described above, according to the
method of C. Kaiser et al. (18). Exactly 20 µg of proteins
in the extract was resolved by SDS-10% PAGE for detection of GCN4 or
SDS-15% PAGE for detection of MBF1 and then subjected to Western blot analyses as described previously (26). The bound antibodies were visualized by using a Super Signal CL-HRP substrate system (Pierce). Antisera against GCN4 and MBF1 were gifts from A. G. Hinnebusch and M. Jindra, respectively. The antiserum against GCN4 was
used after 250-fold dilution followed by incubation with a membrane
that had been blotted with proteins in the extract from the
gcn4 strain. The antiserum against MBF1 was used after 10,000-fold dilution.
Nucleotide sequence accession number.
The nucleotide
sequence encoding a part of yMBF1 has been deposited in the
GenBank/EMBL/DDBJ databases as an expressed sequence tag of unknown
function with accession no. T38224.
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RESULTS AND DISCUSSION |
Yeast MBF1 is essential for transcriptional activation of
HIS3.
Curiously, the partial yMBF1 sequence was not
recognized as an open reading frame in the yeast genome sequence
(10). The nucleotide sequence of the complete open reading
frame has been determined, and the deduced amino acid sequence has been
compared with MBF1 sequences of other organisms (34). yMBF1
has 43% amino acid identity with the Drosophila
counterpart. To investigate the role of yMBF1 in vivo, we inactivated
the MBF1 locus by one-step gene replacement. The disruptant
(the mbf1 strain) was viable (Fig.
1A) and able to grow on galactose,
sucrose, or inositol-free media (data not shown), indicating that yMBF1
is not a general transcription factor. But the mbf1
strain was sensitive to AT, an inhibitor of the HIS3 gene
product (Fig. 1A). This sensitivity was suppressed by introducing a
yMBF1 expression plasmid, pMBF1. We also examined the level
of HIS3 mRNA by quantitative S1 nuclease mapping (Fig. 1B).
Upon histidine starvation, no activation of HIS3 mRNA
synthesis was detected in the mbf1 strain, and this phenotype was the same as that observed for a GCN4
disruptant (gcn4 strain). Activation of HIS3
transcription was restored when pMBF1 was introduced into
the mbf1 strain. These results clearly show that yMBF1 is
essential for transcriptional activation of the HIS3 gene.
The mbf1 strain was also defective in transcriptional activation of the HIS5 gene upon histidine starvation (data
not shown), suggesting that yMBF1 plays a role in GCN4-dependent
activation.
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FIG. 1.
Phenotype of the mbf1 strain. (A) AT
sensitivity. Cells of the wild type (WT), the gcn4
strain, the mbf1 strain, and the mbf1
strain transformed with pMBF1 were grown on plates in the
presence or absence of 10 mM AT for 3 days at 30°C. (B) Effect on
HIS3 transcription. Total RNA from the four strains grown
under the histidine-starved conditions was used for S1 nuclease
analyses of HIS3 and DED1 transcripts.
Constitutive HIS3 transcripts initiate from the +1, +13, and
+22 sites, whereas GCN4-activated transcripts initiate preferentially
from the +13 sites; DED1 is not affected by GCN4 and serves
as an internal control (33). (C) Expression of
GCN4 in the mbf1 strain. The levels of GCN4 in
the four strains were estimated by Western analysis with an anti-GCN4
antiserum.
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GCN4-MBF1 interaction is necessary for GCN4-dependent activation of
HIS3.
Because GCN4 is required for activation of the
HIS3 gene (33), we examined expression of
GCN4 in the mbf1 strain. Virtually the same
levels of GCN4 were observed in the wild type, the mbf1 strain, and the mbf1 strain harboring pMBF1
when Western blots of 20 µg of proteins in each yeast extract were
probed with the antiserum against GCN4 (Fig. 1C). Under these
conditions, signal intensity increased with protein loading up to at
least 20 µg (data not shown). No GCN4 protein was detected with the
antiserum in the gcn4 strain, which was used as a
control.
We next analyzed interaction between yMBF1 and GCN4. GST pull-down
assays of bacterially expressed and purified proteins showed
that GCN4
binds directly to yMBF1 (Fig.
2A; compare
lanes 2 and
3). In this and all subsequent GST pull-down assays,
addition
of 50 µg of ethidium bromide per ml to the binding mixture
did
not affect the interactions, suggesting that the observed
interactions
were not due to bridging through contaminating nucleic
acid (data
not shown). The DNA-binding domain of GCN4 (bZIP; amino acid
positions
222 to 281) is necessary and sufficient for the binding (Fig.
2A; compare lanes 6 and 7 with lanes 9 and 10). No binding occurred
in
the absence of Mg
2+, and the complex that formed in the
presence of Mg
2+ dissociated immediately upon removal of
Mg
2+ (data not shown). Deletion of a central region (amino
acid positions
41 to 119) of yMBF1 (yMBF1
41-119) abolished the
binding of GCN4
(Fig.
2A, lanes 4 and 11). The corresponding region of
insect
MBF1 has been shown to be essential for the binding of FTZ-F1
(
34). Furthermore, we observed a significant increase in the
DNA binding of GCN4 upon addition of purified yMBF1 to gel mobility
shift assay mixtures (Fig.
2B). Competition with unlabeled
oligonucleotide
carrying the wild-type or mutant GCN4-binding site
revealed that
the shifted bands represent the GCN4-DNA complex (data
not shown).
When we used yMBF1
41-119, which does not interact with
GCN4,
no increase in the GCN4 binding to DNA was detectable (Fig.
2B).
Essentially the same results were obtained when we used GCN4bZIP
in
place of intact GCN4 (data not shown). The effect appears to
be
specific to GCN4, as yMBF1 did not increase the DNA binding
of other
sequence-specific factors (for example, see Fig.
6 for
TBP binding).
Because the mixture contained 5 mg of BSA per ml,
it is highly unlikely
that the yMBF1 simply prevented either GCN4
denaturation or sticking of
GCN4 to the test tube. Accumulation,
but not supershift, of the
GCN4-DNA complex was detected upon
addition of MBF1, as observed for
the FTZ-F1-DNA complex (
34).
This is due to immediate
dissociation of the GCN4-MBF1 complex
in the electrophoresis buffer
without Mg
2+. No qualitative changes were observed in the
DNase I footprinting
patterns of GCN4 upon addition of yMBF1 (data not
shown). Therefore,
we surmise that the contact of yMBF1 with the
DNA-binding domain
of GCN4 induces some conformational change in GCN4,
allowing increased
binding to DNA.
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FIG. 2.
Direct interaction between yMBF1 and GCN4. (A) GST
pull-down assay for interaction of yMBF1 with GCN4. Bacterially
expressed and purified HA epitope-tagged GCN4 (lanes 2 to 4),
GCN4bZIP (lanes 6 and 7), or GCN4bZIP (lanes 9 to 11) (100 ng each)
was incubated with 200 ng of either GST (lanes 2, 6, and 9), GST-yMBF1
(lanes 3, 7, and 10), or GST-yMBF141-119 (lanes 4 and 11). The
bound GCN4 was electrophoresed by SDS-10% PAGE, and its immunoblot
was probed with an anti-HA antibody. Lanes 1, 5, and 8: 1/10 the input
GCN4 or its derivatives. (B) yMBF1 increases GCN4 binding to DNA. The
binding of bacterially expressed and purified GCN4 to a
32P-labeled oligonucleotide carrying the GCN4 binding site
of HIS3 was analyzed by an electrophoresis mobility shift
assay in the presence or absence of the indicated amounts of purified
His-tagged yMBF1 or yMBF141-119.
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Interestingly, a part of the basic region in the GCN4 bZIP motif,
RRSRARKLQRMKQ (underlined residues in Fig.
3), has homology
with the C-terminal half
of the FTZ-F1 box KRDRARKLQVMRQ (
36)
to which insect MBF1
binds (
34) (identity of 9 amino acids and
similarity of 11 amino acids of 13 residues). Therefore, we prepared
four mutant forms
of GCN4 in which each of the four arginine residues
within the
homologous region was replaced with alanine (R240A,
R241A, R243A, and
R245A). We also prepared two point mutants carrying
replacements
outside the homologous region (N235A and A238G).
Bacterially expressed
and purified mutant proteins migrated during
SDS-PAGE with essentially
the same mobility as the wild-type GCN4
(data not shown). GST
interaction assays with these purified proteins
showed that all four
mutations within the homologous region reduced
the binding to yMBF1
while the two point mutations outside the
homologous region did not
diminish but rather enhanced the binding
(Fig.
3, column III). Gel
mobility shift assays with purified
proteins revealed that all six
mutations reduced the DNA binding
to various degrees (Fig.
3, column
II). In the presence of 200
ng of yMBF1, the relative DNA-binding
activities of the mutants
carrying R241A and R245A were higher than
those of the other mutants
(Fig.
3, column II).
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FIG. 3.
Genetic interaction between yMBF1 and the basic region
of GCN4. Amino acid sequences of the basic region of wild-type GCN4
(WT) and its point mutants are shown. (Column I) AT sensitivity of GCN4
mutants. Indicated GCN4 derivatives were expressed in the
gcn4 strain. Approximately 105 cells were
spotted onto plates in the presence or absence of 20 mM AT and
incubated for 3 days at 30°C. (Column II) DNA-binding activities of
bacterially expressed and purified GCN4 mutants in the presence or
absence of 200 ng of purified yMBF1 as determined by electrophoresis
mobility shift assays. Band intensities of complexes of GCN4 mutant
forms with DNA were quantitated with a Fuji BAS-2000II bioimage
analyzer, and the levels relative to that of the WT are shown. (Column
III) yMBF1-binding activities of GCN4 mutants analyzed by GST assays.
Band intensities of GCN4 mutant forms on Western blots were quantitated
with a Bio Image advanced quantifier, and the levels relative to that
of the WT are shown. (Column IV) Suppression of gcn4
mutations by overexpression of yMBF1. Strains expressing the indicated
GCN4 derivatives with or without pYEMBF1 were assessed for
HIS3 and DED1 transcripts by S1 nuclease
analyses. Relative levels of the HIS3 + 13 transcript
normalized to the levels of the DED1 internal control after
subtraction of the basal level in the gcn4 strain are
shown.
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gcn4 cells expressing any of these mutant constructs were
sensitive to AT (Fig.
3, column I) and defective in transcriptional
activation of the
HIS3 gene upon histidine starvation (Fig.
3,
column IV). The mutant proteins were as stable as the wild-type
GCN4
in vivo (Fig.
4). Overexpression of yMBF1
enhanced the transcription
of
HIS3 mRNA in mutants carrying
R241A and R245A but not in those
carrying R240A and R243A (Fig.
3,
column IV). The sensitivities
to 20 mM AT of the R241A and R245A
mutants but not those of the
R240A and R243A mutants were suppressed by
overexpression of yMBF1,
albeit partially in the mutant carrying R245A
(data not shown).
These in vivo data are consistent with the relatively
high levels
of DNA binding of R241A and R245A in the presence of excess
yMBF1
(Fig.
3 column II). It is likely that arginine residues at 241
and 245 constitute a binding surface for yMBF1, whereas arginine
residues at 240 and 243, on the opposite surface of the
helix,
are
known to face DNA (
7). These data suggest that the binding
of yMBF1 to the basic region of GCN4 is necessary for GCN4-dependent
activation of the
HIS3 gene.
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FIG. 4.
Stability of GCN4 mutant proteins in yeast. The levels
of wild-type GCN4 (WT) and the mutant forms in 20 µg of proteins of
each yeast cell extract were estimated by Western analysis with the
anti-GCN4 antiserum.
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MBF1-TBP interaction is required for transcriptional activation of
HIS3.
Next, we analyzed interaction between yMBF1 and yTBP.
GST pull-down assays with bacterially expressed and purified proteins showed that yMBF1 comes into direct contact with yTBP (Fig.
5A; compare lanes 2 and 3). Like that of
insect MBF1 (34), the central region of yMBF1 (amino acid
positions 41 to 119) is essential for the TBP binding (data not shown).
The yMBF1-yTBP interaction was confirmed by retardation of a yTBP-TATA
element complex upon addition of purified yMBF1 in gel mobility shift
assays (Fig. 6; compare lanes 2 and 3).
We also found that substitution of alanine for aspartic acid at
position 112, within the highly conserved domain of yMBF1
(34) (D112A), reduced the binding of yTBP (Fig. 5A; compare
lanes 3 and 4), whereas the binding of yTBP to a truncated version of
yMBF1 lacking its N-terminal 40 amino acids (yMBF1NT) remained
unaffected (Fig. 5A; compare lanes 3 and 5). The binding of GCN4 was
not altered by the D112A mutation (Fig. 5B; compare lanes 4 and 5) but
was slightly reduced by the N-terminal deletion of yMBF1 (Fig. 5B;
compare lanes 3 and 4). Both the D112A and mbf1NT mutants
are sensitive to AT (Fig. 5C) and express significantly lower levels of
HIS3 mRNA than the wild type (Fig. 5D). Overexpression of
yTBP enhanced the HIS3 mRNA level in the
mbf1D112A strain (Fig. 5D) and suppressed the AT sensitivity
(data not shown). Neither the HIS3 mRNA level (Fig. 5D) nor
the AT sensitivity (data not shown) of the mbf1NT strain,
used as a negative control, was affected by overexpression of yTBP.
These mutant MBF1 proteins were as stable as the wild-type yMBF1 in
vivo (Fig. 5E). These results indicate that the yMBF1-yTBP interaction
is required for transcriptional activation of the HIS3 gene.
Expression of GAL4-MBF1 or LexA-MBF1 fusion protein caused severe
growth inhibition in yeast. This inhibition was alleviated by
overexpression of TBP (34a). These results also suggest that
MBF1 and TBP interact in yeast.
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FIG. 5.
Interaction between yMBF1 and yTBP in vitro and in vivo.
(A) GST pull-down assays for binding of yMBF1, the D112A protein, and
yMBF1NT to yTBP. Bacterially expressed and purified yTBP was
incubated with either GST (lane 2), GST-yMBF1 (lane 3), GST-D112A
protein (lane 4), or GST-yMBF1NT (lane 5). The bound yTBP was
separated by SDS-12.5% PAGE and, after transfer, probed with an
anti-yTBP antibody. Lane 1, 1/10 the input yTBP. (B) GST pull-down
assays for binding of the yMBF1 D112A protein and yMBF1NT to GCN4.
Purified HA epitope-tagged GCN4 was incubated with GST or indicated GST
fusion proteins (lanes 2-5), and the bound GCN4 was detected as
described in the legend for Fig. 2. Lane 1, 1/10 the input GCN4. (C)
yMBF1 D112A and NT mutants show AT sensitivity. The
mbf1 strain transformed with pYCMBF1,
pD112A, or pMBF1NT was tested for growth as
described in the legend for Fig. 3. (D) Suppression of mbf1
D112A by overexpression of yTBP. Total RNA from the gcn4
strain or the mbf1 stain harboring the indicated plasmids
was subjected to S1 nuclease analysis. Relative levels of the
HIS3 + 13 transcript are expressed as in Fig. 3. (E)
Stability of yMBF1 mutant proteins in yeast. The levels of wild-type
yMBF1 (lane 1) and its mutant forms, the D112A protein (lane 2) and
yMBF1NT (lane 3), in 20 µg of proteins of each yeast cell extract
were estimated by Western analysis with an anti-MBF1 antiserum.
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FIG. 6.
yTBP, yMBF1, and GCN4bZIP form a complex on a TATA
element. 32P-labeled oligonucleotide bearing the TATA
element of the HIS3 gene was incubated with various
combinations of bacterially expressed and purified proteins. When
indicated, the incubation mixture contained 10 ng of yTBP, 100 ng of
yMBF1, 100 ng of yMBF1-D112A, and/or 100 ng of GCN4bZIP. The samples in
lanes 1 to 7 were run on the same gel; those in lanes 8 to 10 were run
on another gel. A faint band above the yTBP-TATA element complex
appears to be a nonspecific complex, as it was competed out with a
mutant TATA element carrying GCGC sequence in place of TATA, and hence
we ignored it.
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Formation of a TBP-MBF1-GCN4 ternary complex in vitro.
As
described above, when yMBF1 was added to gel mobility shift assay
mixtures consisting of yTBP and the TATA element of the HIS3
gene, we observed a supershift of the yTBP-TATA element complex (Fig.
6; compare lanes 2 and 3). Such supershift was not detectable upon
addition of yMBF1-D112A (Fig. 6; compare lanes 8 to 10). This result
confirms the weak nature of the interaction of yMBF1-D112A and TBP.
Upon further addition of the DNA-binding domain of GCN4 (GCN4bZIP),
further retardation of the supershifted complex was detected (Fig. 6;
compare lanes 3 and 4). The retardation was observed only in the
presence of yMBF1 and not in its absence (Fig. 6; compare lanes 4 and
5). Similar results were obtained when we used intact GCN4 instead of
GCN4bZIP (data not shown). These results suggest formation of a
yTBP-yMBF1-GCN4 ternary complex on the TATA element. We were unable to
use the anti-GCN4 antiserum or the anti-HA antibody for detection of
GCN4 in the ternary complex by supershift, because neither of these
antibodies supershifted any complex, even the GCN4-DNA complex (data
not shown).
Based on these biochemical and genetic data, we conclude that yMBF1
acts as a coactivator of GCN4 by tethering TBP to the
DNA-binding
region of GCN4 (Fig.
7).
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FIG. 7.
Model for GCN4-dependent activation through yMBF1. yMBF1
serves as a coactivator of GCN4 by tethering TBP to the DNA-binding
domain of GCN4.
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Recruitment of TBP through MBF1 is rate limiting in GCN4-dependent
activation.
When TBP is artificially recruited by covalent
attachment to a promoter-bound protein, an activation domain is no
longer required for transcription (6, 21, 38). It is
possible that the recruitment of TBP by yMBF1 requires the aid of the
activation domain because the protein-protein interaction for tethering
of TBP is not as firm as the covalent linkage of TBP. In agreement with
this idea, transcription of the HIS3 gene in the absence of
the major activation domain of GCN4 was partially activated by
overexpression of yMBF1 but not by overexpression of D112A protein
(Fig. 8). GCN4AD retains a residual
activation domain near its N terminus (16). It is possible
that the observed suppression may be due to enhanced expression of
GCN4AD by overexpression of yMBF1. However, Western analysis of
yeast cell extracts showed that the expression of GCN4AD was not
altered by overexpression of yMBF1 (data not shown). These results
suggest that the recruitment of TBP through MBF1 is a rate-limiting
step in GCN4-dependent activation and hence is a target of regulation.
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|
FIG. 8.
Overexpression of yMBF1 restores activation of
HIS3 to a GCN4 mutant lacking the activation domain. For
evaluation of AT sensitivity, approximately 105 cells of
the gcn4 strain or the gcn4 strain
transformed with the indicated plasmid were grown on plates in the
presence or absence of 2.5 mM AT for 3 days at 30°C. For evaluation
of HIS3 mRNA, total RNA from the gcn4 strain
harboring the indicated plasmids was subjected to S1 nuclease analysis;
relative levels of the HIS3 +13 transcript are expressed as
in Fig. 3.
|
|
The present study illustrates the importance of the basic region within
the bZIP motif of GCN4 for the interaction with yMBF1.
The basic amino
acids relevant to genetic interaction with yMBF1,
R241 and R245, are
conserved at corresponding positions in a subgroup
of bZIP proteins.
This, together with the evolutionary conservation
of MBF1, raises the
possibility that MBF1 serves as a coactivator
for a wider spectrum of
bZIP proteins. The human T-cell leukemia
virus transactivator Tax has
been shown to interact with the basic
region of certain bZIP proteins
and to increase the DNA binding
of these proteins (
2,
28).
Although MBF1 does not have apparent
sequence homology with Tax, the
two may carry out their functions
through a common mechanism.
| |
ACKNOWLEDGMENTS |
We are grateful to K. Struhl for yeast strain KY803 and
YCp88-GCN4, M. Horikoshi for yTBP DNA, R. D. Gietz for yeast
vectors, A. G. Hinnebusch for anti-GCN4 antiserum, and M. Jindra
for anti-MBF1 antiserum. We also thank I. Herskowitz, M. Jindra, J.-I.
Tomizawa, and A. Ishihama for critical reading of the manuscript, H. Mitsuzawa for helpful discussions, and M. Fujino for advice on RNA
isolation.
This work was supported by grants-in-aid for scientific research from
the Ministry of Education, Science, Sports and Culture of Japan. K.T.
was supported by a C.O.E. program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Developmental Genetics, National Institute of Genetics, Mishima,
Shizuoka-ken 411-8540, Japan. Phone: 81-559-81-6771. Fax:
81-559-81-6776. E-mail: shirose{at}lab.nig.ac.jp.
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Abstract
Introduction
