(Received for publication, December 3, 1996, and in revised form, February 25, 1997)
From the Department of Biology, Georgetown
University, Washington, D. C. 20057 and the ¶ Laboratory of
Eukaryotic Gene Regulation, NICHD, National Institutes of Health,
Bethesda, Maryland 20892
Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves down-regulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.
The de novo synthesis of purine nucleotides requires 10 enzymatic steps to form the first purine nucleotide, inosine monophosphate. IMP is converted to either AMP or GMP in two steps. The products of at least 13 genes are required for this synthesis, since mutations in any one of these lead to an adenine requirement (1). Most of these genes encode the biosynthetic enzymes that participate in specific steps of the pathway (ADE1, ADE2, ADE4, ADE5,7, ADE6, ADE8, ADE12, ADE13), others encode enzymes required to produce additional substrates necessary to complete the pathway (ADE3, ASP5), and the function of one gene product is unknown (ADE9). Strains with mutations in two additional genes, BAS1 and BAS2, have a partial adenine requirement (2). In these latter mutants, expression of the biosynthetic enzymes is low, indicating a positive regulatory role for these gene products (3, 4).
Expression of the adenine biosynthetic genes is repressed when cells are grown in the presence of adenine (3-6). This adenine-mediated repression occurs at the transcriptional level (7). ADE gene transcription is unregulated in bas1 or bas2 mutant strains (3). The expression of BAS1 and BAS2 is not regulated by adenine levels (8, 9), however, indicating that adenine repression occurs by down-regulating the activator functions of the BAS1 or BAS2 proteins.
BAS1 and BAS2 were identified as transcriptional activator proteins
required for the basal expression of the HIS4 gene (2). BAS1
binds to DNA using an amino-terminal myb motif (8). This tryptophan-rich motif is repeated three times in the BAS1 protein (8,
10). BAS1 binds to two sites in the HIS4 promoter (8) and to
two sites in each of the promoters for the ADE2 and
ADE5,7 genes (3). The consensus sequence derived from these
six BAS1 binding sites is TGACTC. Interestingly, this hexanucleotide
sequence forms the core of the binding site for GCN4 protein as well,
although flanking nucleotides differently affect the binding affinity
of these two proteins (10, 11). Whereas GCN4 has a preference for the
sequence RRTGACTCATTT (R represents A or G; Ref. 11), none
of the known BAS1 sites or any predicted sites in other ADE gene promoters have either an A nucleotide at the 3 base following the
conserved hexanucleotide core or any other sequence conservation.
BAS2 binds to DNA via an amino-terminal homeodomain that is closely related to the engrailed protein of Drosophila (8). The BAS2 binding site at HIS4 has been mapped to an A + T-rich repeat, TTAA (8). The results of electrophoretic mobility shift assays suggested that BAS2 binds to the ADE2 and ADE5,7 promoters; however, no specific binding sites were identified (3). In addition to participating with BAS1 in transcriptional activation of HIS4 and the ADE genes, BAS2 (also known as PHO2 and GRF10) stimulates transcription of PHO5 (12) and HO (13). High level transcription of HO requires BAS2 in conjunction with SWI5 (13, 14). Binding of BAS2 and SW15 to the HO promoter is cooperative in vitro (13, 15). BAS2 also interacts with PHO4 to induce PHO5 transcription under phosphate starvation conditions (16). Binding of PHO4 is BAS2-dependent and restricted to derepressing conditions as shown by in vivo footprinting experiments (17). There is evidence that BAS2 and PHO4 physically interact at the promoter and that this interaction is regulated by phosphate (18).
Because expression of the genes that are activated by BAS1 and BAS2 is lower when cells are grown in adenine excess, adenine antagonizes the activation function of one or both of these proteins. Excess adenine could inhibit their ability to interact with one another, to bind to DNA, or to interact with components of the transcriptional machinery. Down-regulation of BAS protein function could also occur by covalent modification or through binding of a negative regulator, analogous to binding of the repressor GAL80 to the activator GAL4 (12). Alternatively, a DNA binding repressor might interact with negative control sites in the promoter, as described for the MIG1-TUP1-SSN6 complex that mediates the carbon catabolite repression of GAL genes (12).
To define more clearly the cis- and trans-acting regulatory elements
required for adenine regulation, we performed an extensive analysis of
the ADE5,7 promoter. First, we identified the minimal sequences from ADE5,7 sufficient to confer adenine-regulated
activation (UASADE5,71 function) to
a heterologous CYC1-lacZ reporter lacking its native UASCYC. We then subjected the minimal UASADE5,7 to
extensive mutagenesis by making successive 3-nucleotide substitutions across this element to identify precisely the critical nucleotides contained therein. Our results indicate that of the two BAS1 binding sites at UASADE5,7, the gene-proximal copy is the more critical, being both necessary and sufficient for adenine-regulated promoter activity. Two additional regions were essential for
UASADE5,7 function. One region, located 5 to the critical BAS1
site, is a binding site for ABF1. The other, located 3
to the critical BAS1 site, is an extended A + T-rich element that appears to be a Bas2
binding site. We found that none of the nucleotide substitutions in the
UASADE5,7 led to constitutively derepressed expression, the
phenotype expected from the loss of a repressor binding site. Thus, our
results suggest that the repression of UASADE5,7 function in
adenine-replete cells involves a modification of one of the three
transcriptional activator proteins, BAS1, BAS2, or ABF1, rather than
binding of a repressor.
Saccharomyces cerevisiae
strains AY854 ( GCN4 BAS1 BAS2 ura3-52), AY856 (a
GCN4 bas1-2 BAS2 ura3-52), AY858 (
GCN4 BAS1
bas2-2 ura3-52), AY860 (
GCN4 bas1-2 bas2-2
ura3-52), AY957 (a gcn4 BAS1 BAS2 ura3-52), and
AY862 (a gcn4 bas1-2 bas2-2 ura3-52) were provided
by Kim Arndt (Cold Spring Harbor Laboratories). ElectroMax
Escherichia coli strain DH10B (Life Technologies, Inc.) was
used for transformation (19).
E. coli cells were grown in LB medium supplemented with 100 µg/ml ampicillin. S. cerevisiae cells were grown in synthetic dextrose (SD) medium (20) supplemented with 0.5 mM arginine and 0.3 mM histidine. Adenine was added to 0.15 mM in the cultures grown under repressing conditions.
Oligonucleotides and PlasmidsTable I lists
the oligonucleotides that were used as polymerase chain reaction (PCR)
and sequencing primers for cloning and as probes for DNA binding assays
as described below. Deletion of an XhoI fragment that
contained the UASCYC1 from plasmid pLG699Z (21) generated
pLG699ZXhoI. Plasmid pCM81, a derivative of
pLG699Z
XhoI, contains an oligonucleotide that replaces
the unique XhoI site with adjacent BglII and
XhoI sites (22). Fragments of the ADE5,7 promoter
were generated by the PCR using plasmid pYEADE5,7(5.2R) (23) and the
primers listed in Table I, as described in the relevant figure and
table legends. PCR reactions were performed using Taq
polymerase (Perkin-Elmer) under the following conditions: 30 cycles of
denaturing at 95 °C for 2 min, annealing at 55 °C for 1 min, and
chain extension at 72 °C for 30 s. PCR fragments were digested
with either XhoI or XhoI and BglII,
separated from primers by electrophoresis, purified, and inserted into
pLG699Z
XhoI or pCM81. The ligated products were
transformed into competent E. coli by electroporation (19).
Bacterial transformants were screened for insertions by colony
hybridization (24), by restriction analysis (24), or by whole cell PCR
(25). The nucleotide sequence of the vector-insert junctions was
verified in all constructs by sequence analysis using primer RO-26,
which corresponds to positions
160 to
142 (relative to the ATG
codon) of the CYC1 gene. Plasmids were transformed into
yeast cells using lithium acetate (26) and plated on medium lacking
uracil.
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Plasmid pR116 carries an ADE5-lacZ fusion and was
constructed by first introducing a 3.0-kb BamHI fragment
carrying the E. coli lacZ gene (27) into the
BamHI site located in the ADE5,7 gene of plasmid
pYEADE5,7(5.2R) (23) to produce plasmid pR111. The ~8-kb
SalI to BspEI fragment containing the
ADE5-lacZ fusion from pR111 was subcloned into the
SalI and XmaI sites of a modified form of pRS316,
lacking the -fragment of lacZ, to produce pR116.
Plasmid pR173 contains a fusion of the bacterial glutathione
S-transferase (GST) gene with the full-length coding
sequence of BAS1 and was constructed in three steps. First,
an oligonucleotide duplex formed between RO-97 and RO-98 was inserted
at the BamHI site of pGEX-5X-3 (Pharmacia Biotech Inc.) to
yield pR171. The 5 end of the gene was generated by PCR using
oligonucleotides RO-107 to RO-108 and pCB286 (2). The PCR fragment was
cleaved with NdeI and BglII and ligated into the
NdeI and BamHI sites of pR171 to yield pR172. The
remainder of the BAS1 gene was cloned as a 3.5-kb
BamHI fragment from pCB286, ligated into the same site in
pR172 to yield pR173.
Plasmid pR175, encoding the GST-BAS2 fusion, was constructed in two steps. First, an oligonucleotide duplex formed between RO-103 and RO-104 was inserted at the BamHI and EcoRI sites of pGEX-5X-3 to yield pR174. Plasmid pCB841 (2), which carries BAS2, was partially digested with EcoRI to generate a 2.5-kb fragment that was ligated into the EcoRI site of pR174 to yield pR175. Plasmid M2025 carrying the (His)6-BAS2 fusion was a gift of D. Stillman (15).
Transformants to be assayed for
-galactosidase activities were inoculated in 5 ml of SD medium
supplemented with 0.5 mM arginine, 0.3 mM
histidine and 0.15 mM adenine and cultured for ~42 h.
Each saturated culture was diluted 1:50 in 25 ml of fresh medium, with and without adenine supplementation, and grown for 5 h with
shaking at 30 °C. Cells were harvested by centrifugation and frozen
at
20 °C overnight.
-Galactosidase assays were performed using whole-cell extracts (22).
To generate labeled ADE5,7 DNA
fragments, oligonucleotides RO-41 and RO-136 were end-labeled with
polynucleotide kinase and [-32P]ATP and were separated
from unincorporated label by passage over a G25 spin column. PCR
reactions were performed as described above using the labeled
oligonucleotides as primers and plasmids containing the appropriate
ADE5,7 sequences as templates to amplify the region between
211 and
145 (relative to the ADE5,7 start codon). The
templates were pR224, carrying the wild-type ADE5,7 fragment, or selected plasmids carrying mutated ADE5,7
promoter fragments, constructs numbered 5, 7, 10, 11, 13, 14, 19, 20, 27, 29, 34, 35, 39, and 43 as listed in Table IV. After PCR, a portion of each sample was separated by electrophoresis, and concentrations of
the PCR products were estimated by ethidium bromide staining in
comparison with known concentrations of duplex oligonucleotides of
approximately the same length.
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Oligonucleotides RO-118 and RO-119, encoding ARS1, and
RO-120 and RO-121, encoding a mutant ARS1, were prepared by
end labeling with polynucleotide kinase and [-32P]ATP
(24), removing the unincorporated ATP on G25 spin columns, and
annealing equimolar amounts of each by heating to 65 °C and cooling
slowly to room temperature.
DNA fragments used as competitors were prepared as described above for the radiolabeled PCR probes, omitting the end labeling and purification steps. To generate the fragment carrying mutations in both regions III and IV, RO-136 and FZP20 were used as primers in a PCR reaction. The product of this PCR reaction was then used as template in another round of PCR using RO-136 and FZP21 as primers. The PCR fragment was inserted into pCM81 and subjected to sequence analysis to verify the correct sequence.
Electrophoretic Mobility Shift Assays of DNA Binding by ABF1Yeast strain AY862 was grown to midlog phase and harvested
by centrifugation. Cells were broken using glass beads in buffer A (25 mM Hepes, pH 7.7, 50 mM KCl, 10% glycerol, and
0.5 mM EDTA). Cell lysates were clarified by centrifugation
at 20,000 × g. Labeled DNA probes for electrophoretic
mobility shift assays (EMSAs) were either a 32-base duplex
oligonucleotide containing the ABF1 binding site from ARS1
or radiolabeled PCR fragments containing ADE5,7 sequences,
prepared as described above. DNA binding assays were performed in 20 µl with 10 µg of protein in a whole cell lysate and 10 fmol of DNA
in a buffer containing 25 mM Hepes, pH 7.7, 150 mM KCl, 5 mM MgCl2, 1 µg of
poly(dI·dC), and 1 µg of sonicated calf thymus DNA. Binding
reactions were incubated at room temperature for 30 min and separated
by gel electrophoresis using 6% polyacylamide gels in 22.3 mM Tris borate, pH 8.3, 0.5 mM EDTA. The gel
was pre-electrophoresed for 15 min prior to loading samples. Supershift experiments were performed by adding either preimmune serum or immune
serum raised against partially purified ABF1 to the binding reactions.
The antisera, kindly provided by Bruce Stillman (28), were diluted in
buffer A as indicated in the legend to Fig. 3. Competitions with
unlabeled DNA fragments were performed using wild-type or mutant
ARS1-containing oligonucleotides, or with PCR-derived
fragments of the ADE5,7 promoter prepared as described above.
Electrophoretic Mobility Shift Assays of DNA Binding by BAS1
Bacterial strains containing the GST-BAS1 fusion construct
or the pGEX empty vector were inoculated at a 1:100 dilution from saturated cultures into 50 ml of LB containing ampicillin. Cultures were grown for 2.5 h, and IPTG was added to 0.5 mM for
induction of the GST proteins. Cells were harvested by centrifugation
after an additional 2.5 h of growth, frozen in a dry ice/ethanol
bath, and stored at 80 °C. Pellets were thawed on ice and
resuspended in buffer A containing 10 mM 2-mercaptoethanol
and a mixture of protease inhibitors: 1 µg/ml aprotinin, 0.5 µg/ml
leupeptin, 1 µg/ml antipain, 1 µg/ml chymostatin, and 1 mM phenylmethylsulphonyl fluoride. Cell extracts were
prepared by sonication with four 15-s pulses in an ice water bath and
by three cycles of freezing in a dry ice/ethanol bath and thawing in
water. Cellular debris was removed by centrifugation at 20,000 × g for 15 min. Extracts were loaded onto a glutathione
S-Sepharose column with a 2-ml bed volume equilibrated in
buffer A. GST control and GST-BAS1 fusion proteins were eluted from the
column with 10 mM reduced glutathione in buffer A. Electrophoretic mobility shift assays were performed in 10 µl using
0.25 µg of purified protein and 10 fmol of wild-type or mutant DNA
fragments in a buffer containing 25 mM Hepes, pH 7.7, 50 mM KCl, 0.5 mM EDTA, 1 mM
dithiothreitol, 0.5 µg of poly(dI·dC), and 10% glycerol (8).
Unlabeled DNA fragments were added as competitors in a 200-fold molar
excess. Samples were fractionated by electrophoresis on 6%
polyacrylamide gels, as described above for ABF1 binding.
GST-BAS2 was prepared and used in EMSAs as described above
for GST-BAS1. The (His)6-BAS2 protein was prepared for use
in EMSA experiments as described (13, 15) with the following
modification. Crude cell lysates (1.5 mg) from bacteria transformed
with GST-BAS2 plasmid pM2025 (13) or the empty vector were prepared and
added to 200 µl of Ni2+ charged His-Bind resin (Novagen).
The fraction that eluted with 300 mM imidazole was dialyzed
against buffer: 20 mM Tris-HCl, pH 8.0, 0.5 mM
EDTA, 100 mM NaCl, 0.5 mM dithiothreitol, 10%
glycerol, and the protease inhibitors described above. Bovine serum
albumin as a carrier protein was added to a final concentration of 0.75 mg/ml before storage at 80 °C. Electrophoretic mobility shift assays were performed using 1.6 µl of these purified protein
fractions.
We began our study of adenine regulation by analyzing expression of an ADE5,7-lacZ fusion in various strains grown in the presence and absence of exogenous adenine. We assayed wild-type and mutant strains lacking BAS1, BAS2, or GCN4. We found that ADE5,7 promoter activity under derepressing conditions (without Ade medium) and repressing conditions (with Ade medium) was lost in strains containing the mutant alleles bas1-2 or bas2-2 but was not significantly affected in a strain containing the mutant gcn4 allele (Table II). These results indicated that BAS1 and BAS2 are both essential for transcriptional activation of ADE5,7 under derepressing conditions, and they are consistent with the idea that the adenine repression involves down-regulation of BAS1 or BAS2 (3). In addition, they show that GCN4 is dispensable for ADE5,7 promoter activity and its regulation by adenine under these growth conditions (7).
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The results in Table II suggest that the ability of BAS1
and BAS2 to activate ADE5,7 transcription is completely
inhibited in adenine-replete cells. It was not known, however, whether
ADE5,7 promoter activity is dependent on any additional
positive regulators that might be subject to adenine repression or
whether repression involves a DNA binding repressor. To address these
issues, we set out to identify the cis-acting sequences required for
adenine-regulated promoter activity by determining the smallest DNA
fragment from ADE5,7 sufficient to confer
adenine-repressible UAS function on a CYC1-lacZ reporter
(21). Various fragments of the ADE5,7 promoter were
generated by PCR and inserted ~185 nucleotides upstream of the
CYC1 transcription initiation sites where the UASCYC normally resides (Fig. 1). These constructs were
introduced into four different yeast strains carrying wild-type or
mutated alleles of GCN4, BAS1, and BAS2, and
-galactosidase expression was assayed after growing transformants in
minimal medium containing or lacking adenine. As expected, expression
of
-galactosidase in transformants of each strain bearing the
parental CYC1-lacZ construct lacking UASCYC1 was
very low (~10 units) and essentially unaffected by adenine
supplementation or mutations in any of the regulatory genes (Table
III, line 19). The ADE5,7 promoter fragments
exhibited a wide range of expression from the CYC1 promoter
in the wild-type strain, and in many cases promoter activity was
repressed by adenine in the medium. As seen with the authentic
ADE5,7 promoter (Table II), expression depended upon the
wild-type alleles of BAS1 and BAS2 but not upon
GCN4 (Table III). In addition to these adenine-regulated constructs, promoter fragments with 3
end points at positions
183
(Table III, lines 3 and 7) or at
160 (Table III, lines 2 and 10)
exhibited significant activity that was independent of GCN4, BAS1, and BAS2 and was largely unaffected by adenine in
the medium.
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Fig. 1 presents the -galactosidase activities of the constructs
analyzed in Table III after subtracting the promoter activity that was
independent of BAS1, BAS2, and GCN4 (see Fig. 1 legend). The constructs
containing both the distal and proximal BAS1 binding sites (3)
exhibited the highest expression under derepressing conditions (Fig.
1A, lines 1, 2, 4,
5, and 6). In addition, each of these constructs
showed substantial repression by adenine, with repression ratios
ranging from 8.4 to 44. One notable difference among this group of
constructs was observed in the comparison between pR136 and pR148,
where removal of the 12 nucleotides between
145 and
133 led to a
roughly 5-fold increase in expression under both growth conditions
(compare lines 4 and 5). We attribute this difference to the removal of a negative element that is either located
between positions
133 and
145 or was formed by the novel junction
between position
133 in the ADE5,7 fragment and the CYC1 promoter sequences. Because the magnitude of adenine
repression was not diminished by removal of the
133 to
145
interval, no additional studies were carried out on these sequences.
Truncating the ADE5,7 fragment from position
145 to
160
(compare lines 5 and 6) led to a decrease in
UASADE5,7 function by roughly a factor of 2, suggesting that a
positive element resides within this interval. As shown below, the
145 to
160 interval contains a binding site for BAS2.
The two constructs lacking the gene-proximal BAS1 site but containing
the distal site (Fig. 1A, lines 3 and
7) showed very low unregulated activity, suggesting that the
proximal BAS1 site is critical for UASADE5,7 function. In
accord with this idea, three constructs containing the proximal site
but lacking the distal BAS1 site (lines 8-10) showed
substantial promoter activity that was adenine-repressible. As a group,
these last three constructs showed weaker UASADE5,7 function
than the constructs containing both BAS1 sites, suggesting that the presence of two BAS1 sites confers greater UASADE5,7 function
than occurs with the proximal site alone. As above, removal of
positions 160 to
145 from pR145 decreased UASADE5,7 function, consistent with the presence of a positive element in this
region.
The results obtained with constructs pR151, pR150, and pR149
(lines 11-13) revealed that the region adjacent to the
proximal BAS1 site is not sufficient for high level UASADE5,7 function. The very low expression shown by these constructs suggested that a critical positive element resides between positions 211 and
195. Although they exhibited very low promoter activity under derepressing conditions, it appears that all three constructs are
adenine-repressible.
To confirm the different contributions of the two BAS1 binding sites to
UASADE5,7 function, we introduced point mutations into each
BAS1 site in construct pR134, containing the ADE5,7 promoter
fragment from position 233 to
160. A mutation that alters BAS1
binding site from TGACTC to TGAATTC was
introduced into one or both of the BAS1 sites in pR134. An equivalent
substitution was shown to impair in vitro binding of BAS1 to
its sites in the ADE2 promoter (3). The mutation in the
distal BAS1 binding site reduced expression by a factor of about 4 but
did not completely eliminate adenine regulation (Fig. 1B,
pR137). In contrast, a mutation in the proximal site virtually
eliminated both promoter activity and adenine regulation (pR138). The
doubly mutated construct pR140 also showed very low expression, similar
to pR138. Comparable results were obtained when these point mutations
were introduced singly into pR136, which contains the ADE5,7
fragment extending from position
133 to
233 (Fig. 1B,
lines 5-7). These findings agree with the results of the
deletion analysis in Fig. 1A in showing that the two BAS1
sites are not equivalent; the proximal site is essential for promoter
activity and adenine repression, whereas the distal site only augments
UASADE5,7 function.
Given the absence in the literature of
defined binding sites for BAS2 at ADE5,7 and ADE2
and our identification of a positive element located between the two
BAS1 binding sites, we decided to map all the DNA elements in the
UASADE5,7 critical for promoter function and adenine regulation
by saturation mutagenesis. To accomplish this goal, we introduced
random substitutions into the 22 successive groups of three nucleotides
that span the 67-nucleotide ADE5,7 fragment in pR145. This
construct was chosen because it contains the smallest fragment that
conferred high level promoter activity that was strongly repressed by
adenine (Fig. 1A, line 9). The presence of a
single BAS1 binding site in this construct simplified our analysis of
the minimal cis-acting requirements for UASADE5,7 function. In
all, 59 clustered substitution mutations were assayed for their effects
on -galactosidase expression in BAS1 BAS2 GCN4 strain
AY854, under both adenine-repressing conditions and derepressing
conditions. The results shown in Table IV indicated that
substitutions in four regions, defined by constructs 3-19, 26-36,
39-43, and 47-52, reduced expression from that given by the parental
construct. In all cases, expression was decreased to a greater extent
under derepressing versus repressing conditions, leading to
a reduction in the repression ratio.
Fig. 2 summarizes the results obtained under
derepressing conditions for representative samples taken from Table IV,
normalized to the level of expression seen for the parental wild-type
construct. It can be seen that mutations in three of the four regions
severely impaired expression under derepressing conditions (boxed
sequences I-III), whereas mutations in the fourth region
decreased expression by only about 40-50%. Mutations outside of these
four regions had little (<25%) or no effect on expression. Region I
is defined by mutations in clusters numbered 2-6, spanning positions
208 to
194 (Fig. 2), that lowered expression under derepressing
conditions from 120 units to 6-16 units. These were the only clustered
substitutions mutations that also substantially decreased expression
under repressing conditions, lowering it from 10 units to 3-5 units
(Table IV, lines 3-19). Thus, most of the mutations in region I
reduced the magnitude of adenine repression but did not abolish it,
decreasing the repression ratio from ~7 (measured for the parental
construct) to values of 2-4-fold (Table IV). Region I is coincident
with the interval between
211 and
195 whose deletion led to a
dramatic reduction in UASADE5,7 function. Thus, the results of deletion and substitution mutations agree in suggesting that region I
stimulates UASADE5,7 function by roughly an order of magnitude,
without being absolutely required for regulation by adenine.
The sequence in region I conforms exactly to the consensus binding site
for ABF1 (29) (5-CGTNNNNRRYGAY-3
in which R represents any purine, Y
represents any pyrimidine, and N represents any nucleotide (30)), and
the data in Table IV are consistent with the idea that region I
functions as an ABF1 binding site. Mutations in clusters 2-3 and 5-6
alter the conserved positions in this bipartite consensus sequence and
have a large effect on expression, whereas mutations in cluster 4 alter
the nonconserved positions in the consensus sequence (NNR) and have a
relatively weak effect on promoter activity (Table IV).
Region II is defined by mutations in clusters 10-13, spanning
positions 184 to
173 (Fig. 2), of which the most severe decreased promoter function under derepressing conditions by about 10-fold. There
was little change from the wild-type level under repressing conditions
(Table IV, lines 26-36). A complete loss of adenine regulation
distinguishes the mutations in this region from mutations in the other
three regions. The first two clusters in region II, TGA and CTC, alter
the core sequence of the proximal BAS1 binding site described
previously at ADE5,7 (3) and identified above as being
critical for UASADE5,7 function. As expected, all six
substitutions in these two clusters essentially abolished expression
and adenine repression. Mutations affecting the next two clusters in
region II were also very deleterious, including the substitution of TCA
for GTG at cluster 12 and GGA for TCC at cluster 13 (Fig. 2 and lines
34 and 35 in Table IV). We propose that all of the mutations in the
12-nucleotide sequence comprising region II impair UASADE5,7
function by reducing BAS1 binding to the ADE5,7
promoter.
Region III consists of clusters 15 and 16, spanning 169 to
164
(Fig. 2). The most severe mutations in this region decreased expression
from 120 units to 13-40 units under derepressing conditions but had
little or no effect under repressing conditions (Table IV, lines
39-40, 42, and 43). The sequence and location of this region, TAATAA,
resembles the binding site described for BAS2 at HIS4 (8).
The sequence of region IV comprising clusters 18-20, CAATAATGA, is
related to that of region III except that two of the three TAA repeats
are degenerate. Mutations in region IV have the weakest effects,
decreasing expression under derepressing conditions by 50% or less
(Table IV, lines 47-52). On the basis of their sequence and
juxtaposition 3
to the BAS1 site, we conclude provisionally that
regions III and IV function as strong and weak binding sites,
respectively, for BAS2.
None of the clustered substitutions in the UASADE5,7 led to high, unregulated promoter activity. The greatest level of expression under adenine-repressing conditions was observed for mutations in clusters 19 and 21 (Table IV, lines 50-51 and 53-56). The ~2-fold increase above the repressed values elicited by these mutations clearly does not account for the 6.7-fold repression ratio seen with the parental construct. This result is noteworthy because it suggests that adenine repression does not involve binding of a repressor protein to an upstream repression sequence. If such a repressor existed, its binding site would have to coincide with one of the four positive control sites we identified in UASADE5,7 where, as shown below, ABF1, BAS1, or BAS2 binds. Given that only mutations in the BAS1 site completely abolished adenine regulation, a hypothetical repressor would probably have to compete with BAS1 for binding to its site in region II of UASADE5,7.
Region I in UASADE5,7 Functions as a Binding Site for ABF1Results from the mutational studies described above suggest that region I of UASADE5,7 functions as a binding site for ABF1. ABF1 is an abundant, essential yeast protein first shown to bind DNA at autonomous replicating sequences (ARSs) involved in replication (31). Later, ABF1 was shown to interact with telomeres, the silent mating cassettes, and the promoters of various metabolic genes (29, 30, 32). To demonstrate that region I is a binding site for ABFI, we performed EMSAs to detect DNA binding. Whole cell extracts containing ABF1 were prepared from a gcn4 bas1 bas2 yeast strain (AY862) and incubated with radioactively labeled oligonucleotide probes containing the ABF1 binding site from ARS1 or UASADE5,7. As shown in Fig. 3A, the extract contains a binding activity for both the ARS1 (lane 2) and ADE5,7 (lane 6) oligonucleotides. The addition of a 100-fold molar excess of unlabeled ARS1 oligonucleotides eliminated binding to the labeled ARS1 probe (lane 3) and to a large extent in the case of the ADE5,7 probe (lane 7). A 200-fold molar excess of unlabeled ARS1 oligonucleotide abolished binding to the ADE5,7 probe (data not shown). The tighter binding to ADE5,7 is consistent with the observation that the ABF1 binding site in the ADE5,7 fragment has greater homology to the derived consensus sequence for ABF1 binding than does the ARS1 sequence. The experiment was repeated using oligonucleotides containing a double point mutation in the ARS1 sequence (33) as the competitor DNA. This mutant oligonucleotide failed to compete with either of the labeled probes for protein binding (Fig. 3, lanes 4 and 8). These results strongly suggest that ABF1 can bind in vitro to ADE5,7 DNA.
To demonstrate that the bound protein is ABF1, antibodies against ABF1 (28) were added to the binding reactions containing yeast lysate and labeled ADE5,7 or ARS1 oligonucleotides. The addition of ABF1-antibody, but not preimmune serum, produced a supershift of the DNA-protein complexes, whether the labeled probe contained ADE5,7 sequences (Fig. 3B, lanes 3-5) or ARS1 sequences (data not shown). These results prove that ABF1 binds to the ADE5,7 fragment under these in vitro conditions.
To demonstrate that the mutations in the presumed ABF1 binding site in the UASADE5,7 that impair promoter activation in vivo also reduce ABF1 binding in vitro, we performed EMSA using as probes either the wild-type UASADE5,7 fragment or mutant fragments bearing selected clustered substitutions described in Table IV. Fig. 3C shows that ABF1 binds with nearly equal efficiency to the wild-type ADE5,7 fragment (lanes 1 and 9), to a fragment bearing mutations with no effect on in vivo UASADE5,7 function (lane 5), and to fragments containing mutations that impair UASADE5,7 but lie outside of the presumed ABF1 binding site (lanes 6-8 and 14-16). In contrast, ABF1 did not bind to fragments containing mutations in the conserved positions of the consensus ABF1 binding site in UASADE5,7 (lanes 2, 4, 10, 12, and 13). Moreover, mutations that alter the central nonconserved positions of the consensus binding site had little or no affect on ABF1 binding (lanes 3 and 11), consistent with the known sequence requirements for ABF1 binding. We performed complementary experiments in which mutant and wild-type unlabeled fragments were compared for their ability to compete with labeled wild type ADE5,7 probe for binding of ABF1 (data not shown). These results established a perfect agreement between the effects of mutations in the ABF1 consensus binding site on ABF1 binding in vitro and UASADE5,7 function in vivo. We conclude that ABF1 binds to region I in vivo and is responsible for the positive role of this sequence in the UASADE5,7.
Region II in UASADE5,7 Functions as a 12-Base Pair Binding Site for BAS1We proposed that mutations in region II,
including the six nucleotides located 3 to the TGACTC core, which are
not conserved among different BAS1 binding sites, destroy
UASADE5,7 function by interfering with binding of BAS1. To
verify this, we investigated whether mutations in all four of the
triplets composing region II interfere with BAS1 binding to the
ADE5,7 fragment in vitro. We purified a GST-BAS1
fusion protein expressed in E. coli and used it in EMSA
experiments with wild-type or mutant ADE5,7 probes. As shown
in Fig. 4A, GST-BAS1 bound to the
ADE5,7 fragment (lane 3), whereas GST alone did
not (lane 2). Mutations in clusters 10-13 reduced or
eliminated the ability of GST-BAS1 to bind to the ADE5,7 DNA
(Fig. 4B, lanes 5-8), whereas mutations outside
of region II had no effect on binding (lanes 2-4,
9, and 10). Mutations in the TGACTC core sequence
(lanes 5 and 6) and in the adjacent
hexanucleotide sequence GTGTCC (lanes 7 and 8) reduced or abolished binding of GST-BAS1 to the ADE5,7
probe. Three additional mutations in the GTGTCC sequence (constructs 26, 30, and 32 in Table IV) showed strongly diminished or no in vitro binding of GST-BAS1 (data not shown).
To confirm these results, we compared the ability of unlabeled mutant fragments present in 200-fold molar excess to compete with the labeled wild-type fragment for binding to GST-BAS1. These results, shown in Fig. 4C, completely mirrored those presented in Fig. 4B, in that only the mutations in clusters 10-13 of region II diminished the ability to compete with the wild-type ADE5,7 fragment for binding to GST-BAS1. Moreover, the mutations in the TGACTC core sequence (lanes 10 and 11) had a greater effect than did those in the adjacent hexanucleotide sequence (lanes 12 and 13). We conclude that region II comprises a 12-bp binding site for BAS1 and that all of the mutations in this region reduce UASADE5,7 function by decreasing BAS1 binding to the promoter.
Evidence That Regions III and IV in UASADE5,7 Function as BAS2 Binding SitesBased on their A + T-rich sequence and
location 3 to the BAS1 binding site, we suggested above that regions
III and IV are binding sites for BAS2. To test this idea, we carried
out EMSA experiments using a GST-BAS2 fusion protein expressed in
E. coli to determine whether mutations in clusters 15 or 16 reduced binding of BAS2 to the ADE5,7 fragment. GST-BAS2
bound specifically to the wild-type ADE5,7 fragment (Fig.
4A, lane 4), whereas mutations in cluster 15 or
16 of region III significantly reduced binding of this interaction
(Fig. 5A, lanes 1, 5,
and 6). In an effort to verify these results, we carried out
EMSA experiments comparing wild-type and mutant competitors using a
purified polyhistidine-tagged form of BAS2 expressed in E. coli (13, 15). We saw small reductions in the ability of fragments
containing mutations in clusters 15 and 16 or clusters 18 and 19 relative to the wild-type ADE5,7 fragment to compete with
the labeled wild-type probe for (His)6-BAS2 binding (data
not shown). We then combined these mutations in the same fragment and
compared it with the wild-type fragment for ability to compete. As
shown in Fig. 5B, we found that significantly greater
amounts of the fragment containing mutations in clusters 15-19 were
required to achieve the same degree of competition for
(His)6-BAS2 binding to ADE5,7 DNA. These results
support the idea that sequences in regions III and IV function as
binding sites for BAS2. The fact that mutations in clusters 15 and 16 have only modest effects on BAS2 binding in vitro but
substantially reduce UASADE5,7 function in vivo
suggests that the in vitro binding reactions do not
duplicate some important aspect of the in vivo interaction
of BAS2 with the ADE5,7 promoter.
Our results define
the minimum promoter elements required for adenine-regulated
transcription of ADE5,7 and provide a detailed picture of
the spatial relationships among the DNA-binding proteins that interact
with these sequences. These results provide the first complete
description of a UAS from an adenine biosynthetic gene. Wild-type
promoter activity and adenine repression require (i) two binding sites
for BAS1 that are separated in the promoter by about 20 nucleotides,
(ii) a single binding site for ABF1 located immediately 3 to the
distal BAS1 site, and (iii) two A + T-rich elements located immediately
3
to the proximal BAS1 site to which monomers of BAS2 appear to bind,
all within a region of only ~65 bp (Fig. 6). A 52-bp
fragment containing a single binding site for each protein was
sufficient to confer adenine-repressible activation, UASADE5,7
function, on the CYC1 basal promoter.
The conclusion that regions I and II function in UASADE5,7 as
binding sites for ABF1 and BAS1, respectively, is based on the strong
correlation between the effects of mutations in these sequences on
protein binding in vitro and on UASADE5,7 function
in vivo. The results of clustered substitution mutagenesis of region II provides in vivo evidence that BAS1 recognizes
between 4 and 6 bp 3 to the highly conserved TGACTC core element
present in all known BAS1 sites. Similarly, Høvring et al.
(10) showed that BAS1 recognizes the G residues on both strands of the
sequence CGG located immediately 3
to the TGACTC core at the BAS1
binding site in the HIS4 promoter. This particular sequence
is not conserved in either of the BAS1 binding sites at
ADE5,7 or ADE2. Based on our findings, we suggest
that sequence differences in the 4-6 bp on the 3
side of the TGACTC
core element lead to differences in BAS1 binding affinity.
Our proposal that regions III and IV function as BAS2 binding sites is
reasonable given that BAS2 is required for ADE5,7 promoter activation and that these are the only cis-acting elements in addition
to the BAS1 and ABF1 sites that are critical for UASADE5,7 function in vivo. The BAS2 binding sites have not been
identified in any other ADE gene promoter; however, the high
A + T content of the region III and IV sequences and their location 3
of the proximal BAS1 binding site are both reminiscent of the
juxtaposed BAS1 and BAS2 binding sites at HIS4 (8). The
identification of the BAS2 binding sites at ADE5,7 is not
definitive because mutations in regions III and IV that greatly
impaired activation in vivo had relatively small effects on
DNA binding in vitro, and it was necessary to combine
multiple substitutions in these sequences before achieving a
significant reduction in BAS2 binding. One possible explanation for
these results is that purified BAS2 protein may not bind to
ADE5,7 DNA with high sequence specificity. Perhaps specific
binding of BAS2 to regions III and IV is stabilized in vivo
by its interaction with BAS1 bound at the adjacent sequences at
positions
184 to
173 (Fig. 6). As discussed below, we recently obtained strong evidence for complex formation by BAS1 and BAS2 (9).
Although no cooperativity was detected between BAS1 and BAS2 in binding
to the HIS4 promoter (8), the BAS2 site defined at
HIS4 (repeats of TTAA; Ref. 8) differs from that proposed here for ADE5,7 (TAATAA and CAATAA). It is possible that
BAS2 has a greater affinity for the HIS4 sequences and does
not require cooperativity with BAS1 to bind there with high
specificity.
We recently obtained evidence for interactions between BAS1 and BAS2 by showing that a LexA-BAS1 fusion protein can recruit BAS2 to a promoter containing lexA operator sequences (9). This interaction at the lexA operator occurred even when the DNA binding activity of BAS2 was destroyed by mutations in its homeodomain. These mutations impaired activation of ADE5,7 transcription, however, indicating that the DNA binding activity of BAS2 was required for its binding to the native ADE5,7 promoter. To explain these observations, we propose that BAS1 cannot bind to the ADE5,7 promoter as effectively as LexA-BAS1 binds to the lexA operator and that an interaction between BAS1 and BAS2 is required for these proteins to bind with high affinity to their adjacent sites at ADE5,7.
We propose that the BAS2 binding sites at ADE5,7 are relatively weak and that cooperativity with BAS1 is required for stable binding of BAS2 to regions III and IV. Similarly, stable binding of BAS1 at the ADE5,7 proximal site depends on cooperativity with BAS2. In contrast, both proteins can interact more effectively with their respective binding sites at HIS4, and cooperativity is not required for promoter occupancy.
Possible Functions for ABF1 and the Distal BAS1 Binding Site in the ADE5,7 PromoterOur results showed that the two BAS1 binding
sites in the ADE5,7 promoter are not equivalent. Mutations
in the distal site led to an approximately 6-fold loss in expression
but no obvious impairment of adenine regulation. In contrast, the
proximal BAS1 binding site was absolutely required for promoter
activity and regulation. Given that BAS1 and BAS2 must interact to form
a potent activation complex (9), we propose that juxtaposition of BAS2 binding sites on the 3 side of the proximal BAS1 site allows formation
of the functional BAS1-BAS2 complex. The fact that the distal BAS1 site
is flanked by an ABF1 site rather than a BAS2 binding site may explain
the failure of the latter to support UASADE5,7 function without
the proximal site. In addition to ADE5,7, the genes
ADE2, ADE4, ADE1, and ADE8 have two TGACTC core
sequences that are potential BAS1 binding sites, and it was shown that
BAS1 binds in vitro to both sites at ADE2. The
proximal TGACTC sequence is more important for activation at
ADE2 (3, 4, 6), whereas the distal site is more critical at
ADE4 (34). It will be interesting to learn whether the more
potent sites at these genes are uniquely flanked by BAS2 binding sites, in the manner we suggest for ADE5,7.
At ADE5,7, the two BAS1 binding sites are separated by 33 nucleotides, which is 3.1 helical turns. The two TGACTC core sequences in the other ADE genes are also separated by an integral number of helical turns (with the exception of the ADE1 gene), placing the BAS1 binding sites on the same face of the DNA helix. McBroom and Sadowski (35) have shown that ABF1 is able to bend DNA 120° around itself. If this occurred at the ADE5,7 promoter, then binding of ABF1 could bring the distal and proximal BAS1 sites into close proximity. Perhaps the distal BAS1 site has a higher affinity for BAS1, and the bend in the DNA introduced by ABF1 allows BAS1 bound at the distal site to stabilize binding of the BAS1-BAS2 complex to the proximal site. Alternatively, the bend in the DNA might allow BAS1 bound at the distal site to cooperate with the BAS1-BAS2 complex in promoting assembly of the transcription initiation complex, although it is incapable of carrying out this function alone.
Because the distal BAS1 site is less important than the ABF1 site for high level activation, ABF1 must perform a function distinct from promoting interactions between BAS1 molecules bound at the distal and proximal sites at ADE5,7. Perhaps ABF1 is required to mark the region as a promoter, and by bending the DNA keeps UASADE5,7 free of nucleosomes. It has been proposed that RAP1 binding in the vicinity of BAS1 and BAS2 binding sites performs precisely this function in the HIS4 promoter (36). The proximity of the distal BAS1 site and the ABF1 site at ADE5,7 (Fig. 6) raises the possibility that binding of BAS1 and ABF1 is mutually exclusive. If so, ABF1 may be displaced by binding of BAS1 to the distal site in the course of assembling the transcription initiation complex. Additional in vitro studies using purified BAS1, BAS2, and ABF1 will be required to test this idea and to address the possibility that BAS1-BAS1, BAS1-BAS2, or BAS2-BAS2 interactions stabilize the binding of these proteins to UASADE5,7.
The Mechanism of Adenine Repression of BAS1 and BAS2 FunctionOur mutational analysis provided no evidence for a DNA binding repressor that would mediate adenine repression of ADE5,7 transcription. Mutations in the binding site for such a repressor would be expected to produce constitutively derepressed expression, whereas all of the mutations we generated led to reduced UASADE5,7 function. While it is possible that a repressor protein binds to UASADE5,7 with the same sequence requirements as ABF1, BAS1, or BAS2, this seems unlikely. Moreover, the fact that only mutations in the BAS1 site completely abolished adenine repression probably indicates that the hypothetical repressor would have to compete with BAS1 for binding to its proximal site. A simpler explanation for our findings is that adenine regulation occurs by covalent modification of BAS1 or BAS2, or the binding of a negative regulator to one of these two proteins, that inhibits their ability to interact with DNA or activate transcription. In addition, because BAS2 regulates expression of promoters unrelated to purine biosynthesis, it seems most likely that BAS1 is the target of this negative regulation. Our results using LexA-BAS1 fusions eliminated the possibility that the DNA binding domain of BAS1 is the target of adenine repression (9). Instead, we found that complex formation between BAS1 and BAS2 unmasks the latent activation function of BAS1 and is down-regulated by adenine. It is possible that the activation function of the BAS1-BAS2 complex is also negatively regulated by adenine. Several mechanisms can be proposed to explain how the ability of BAS1 to interact with BAS2 is negatively regulated, including phosphorylation, binding of a negative regulator, or prevention of its entry into the nucleus. Efforts are under way to distinguish between these possibilities and to identify other trans-acting factors that might be involved in this regulatory mechanism.
We thank Nannan Zhu for technical assistance; Gisella Storz for comments on the manuscript; and Kim Arndt, Steven Henikoff, David Stillman, and Bruce Stillman for strains, plasmids, and antibodies.