From the School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, New South Wales 2052, Australia
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ABSTRACT |
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Expression of yeast genes involved in
one-carbon metabolism is controlled by glycine, by
L-methionine, and by nitrogen sources. Here we report
a novel control element containing a core CTTCTT motif mediating the
glycine response, demonstrating that a protein binds this element, that
binding is modulated by tetrahydrofolate, and that folate is required
for the in vivo glycine response. In an heterologous
CYC1 promoter the region needed for the glycine response of
GCV2 (encoding the P-subunit of glycine decarboxylase) mediated repression that was relieved by glycine. It was also responsible for L-methionine control but not nitrogen
repression. GCV1 and GCV3 have an homologous
region in their promoters. The GCV1 region conferred a
glycine response on an heterologous promoter acting as a repressor or
activator depending on promoter context. A protein was identified
that bound to the glycine regulatory regions of GCV1
and GCV2 only if the CTTCTT motif was
intact. This protein protected a 17-base pair
CATCN7CTTCTT region of GCV2 that is conserved
between GCV1 and GCV2. Protein binding was
increased by tetrahydrofolate, and use of a fol1 deletion
mutant indicated the involvement of a folate in the in vivo
glycine response. Tetrahydrofolate or a derivative may act as a ligand
for the transcription factor controlling expression of one-carbon
metabolism genes.
The glycine cleavage system (EC 2.1.2.10), also called the glycine
decarboxylase complex (GDC),1
is an important component of one-carbon metabolism that is integral to
the metabolism of purines, choline, thymidylate, and pantothenate and
the biogenesis of methyl groups (1). It is located in the mitochondrial
inner membrane of animals (2) and plants (3) and in the cytosol of
bacteria (4), where it catalyzes the reversible conversion of glycine
and NAD+ to CO2,
NH4+,
5,10-CH2-H4folate, and NADH (5).
The GDC is composed of four subunits designated P- (pyridoxal phosphate
containing), H- (lipoic acid containing carrier), L- (lipoamide
dehydrogenase), and T- (tetrahydrofolate-requiring) proteins. The roles
of these proteins in the reaction mechanism have been described by
Hiraga and Kikuchi (6). Genes encoding the subunits of the GDC have
been identified from many organisms; in Saccharomyces cerevisiae
GCV1 encodes the T-protein (7), GCV2 the P-protein (8),
GCV3 the H-protein (9) and LPD1 the L-protein
(10). Although the first three genes are unique to the GDC complex, the
LPD1 gene product acts in several other complexes including
pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and a branched
chain oxo-acid dehydrogenase (11, 12).
In most organisms, a major source of one-carbon units is the C-3 of
serine, which is transferred to H4folate in a reversible reaction catalyzed by serine hydroxymethyltransferase generating 5,10-CH2-H4folate and glycine. In S. cerevisiae there are two other significant pathways for generation
of one-carbon units: from glycine via the GDC (13) or from formate via
the cytoplasmic C1-tetrahydrofolate synthase (a
trifunctional polypeptide encoded by ADE3) (14, 15). The GDC
serves an important role in balancing cellular requirements for glycine
and one-carbon units. In S. cerevisiae for example, glycine
can substitute for the serine requirement in a ser1 strain
in which synthesis of serine from glycolytic intermediates is blocked
(16). In this mutant the GDC cleaves some of the glycine to
CO2 and 5,10-CH2-H4folate, providing the one-carbon units required for other reactions in the
cell. Glycine can also serve as a source of nitrogen in the absence of
other more readily metabolized nitrogen sources (17).
In yeast the expression of GCV1, GCV2, and GCV3
is regulated by glycine (7-9) In Escherichia coli
expression of the gcv operon encoding the glycine cleavage
system is induced by glycine and repressed by purines (18), whereas a
mixture of one-carbon metabolites has been reported to repress the
yeast GCV3 gene (9). GCV2 expression is also
greatly reduced in cells grown in rich medium or in good nitrogen
sources compared with defined minimal medium. Because glycine can act
as a poor nitrogen source, expression of the GCV genes may
be subject to some form of nitrogen catabolite repression (19, 20)
mediated by readily assimilated nitrogen sources such as
L-glutamine and L-asparagine.
Here we report a detailed characterization of the molecular mechanism
of GCV2 regulation by glycine and show that the gene is also
subject to control by L-methionine and nitrogen source. The
elements in the promoter of GCV2 responsible for glycine and L-methionine regulation have been identified by deletion
analysis and footprinting, and the gene was shown to be negatively
regulated by repression, which is relieved in the presence of glycine.
These elements act negatively in a heterologous promoter to bring it under glycine control. Similar elements have been identified in the
GCV1 and GCV3 genes.
The control region of the GCV2 promoter has been found to
bind a protein that is present in yeast nuclear extracts or in
heparin-Sepharose purified cell extracts, and a similar complex is
formed with the putative GCV1 control region. The formation
of the complex seen between a protein and the promoter has been shown
to be responsive to H4folate in vitro. Use of a
fol1 deletion mutant, which is inhibited in
H4folate biosynthesis, indicated the need of a folate for
the glycine response in vivo, indicating that
glycine-specific control may be mediated by monitoring the balance of
one-carbon intermediates present in the cell at the folate level.
Materials--
Amino acids, 2-mercaptoethanol, poly(dI-dC),
folic acid, sodium tetrahydrofolate (H4folate), and folinic
acid (5-formyltetrahydrofolate) were obtained from Sigma. Sodium
tetrahydrofolate stock solution (70 mM) was prepared by
adding H4folate to 1 M 2-mercaptoethanol. The
solution was adjusted to pH 7.0 with 2 N NaOH. The solution was bubbled with argon, and the tubes remained sealed until used. [32P]dATP/dCTP (3000 Ci/mmol) was obtained from DuPont.
Restriction and modifying enzymes were obtained from New England
BioLabs or Boehringer Mannheim. Taq polymerase was from
Perkin-Elmer. All other materials were of high quality and obtained
from various commercial vendors.
Strains and Media--
E. coli strain JM101 was used
as host for plasmids. Yeast strain BWG1-7A (MATa
ade1-100 his4-519 leu2-3, 112 ura3-52) was from Dr.
Leonard Guarente, and YUG1 (MATa ura3-52
leu2-3 trp1-289 his-delta1, fol1::KanMX4) was
from Dr. Johannes Hegemann. The yeast media used for the strain
have been described previously (8). Auxotrophic requirements were added
at a concentration of 40 mg/liter. Minimal media with an amino acid as
the sole nitrogen source (GLYmin, GLNmin, ASNmin, or PROmin) consisted
of 2% (w/v) glucose, 0.17% yeast nitrogen base (Difco), and 1.5%
(w/v) amino acid as the sole nitrogen source. (GLN + ASN + NH4+)min was composed of equal
proportions of GLNmin, ASNmin, and Dmin. Standard methods were used for
the growth and manipulation of the yeast cells (21).
DNA Manipulation and Yeast Transformation--
Standard
recombinant DNA and yeast genetic techniques were used (21, 22). The
integrative constructs were based on the S. cerevisiae/E.
coli shuttle vectors (YIp358, YIp356, and YIp357R) carrying the
URA3 selectable marker gene (23) and linearized within the
URA3 sequence with StuI before transformation to
target integration to the ura3 locus of strain BWG1-7A.
Yeast transformations were performed by the lithium acetate procedure
(24). Each transformant was checked by Southern analysis to ensure
integration of the construct as a single copy at the correct locus.
Construction of the GCV1-, GCV2-, and GCV3-lacZ Gene Fusion
Plasmids--
Plasmid pGSD2.5 (8) containing the intact
GCV2 gene was used to prepare plasmid pRH1
(EcoRI/XbaI fragment:
A PCR-based technique was employed to make further deletion constructs
with pGSD2.5 as a template. For each construct, an oligonucleotide
5'-accgtagtaacccttacc-3' (+427 to +444 of GCV2) and a set of
oligonucleotides harboring EcoRI recognition sequences was
used to introduce a restriction site by site-directed mutagenesis. YIp358 as well as the PCR products were cut with
EcoRI/XbaI and ligated to yield the constructs.
The resulting pRH4, pRH5, and pRH6 contained 649-, 611-, and 576-bp
fragments of GCV2, respectively.
Using pRH1 as a template, potential Gcn4p-binding sites (GCN4) at
Window deletion constructs pRH12 and pRH14 were made from pRH7 and
pRH8, respectively. After cutting pRH7 and pRH8 with KpnI, the termini were blunt-ended by the 3' to 5' exonuclease activity of
T4-polymerase. pRH4 was first cut with EcoRI and also
blunt-ended by treatment with Klenow fragment. Then a 650-bp
GCV2 fragment from pRH4 and fragments of pRH7 containing
pRH15 was constructed by inserting annealed oligonucleotides into the
EcoRI site of pRH4. The sequence
The GCV3 gene sequence from
The GCV1 gene cloned into YEp13 was a gift from Dr. Andrew
Bognar. The
The GCV2 fragment Gel Mobility Shift Assay--
Gel mobility shift assays were
performed as described (27). Nuclear protein extracts (28) and protein
extracts using heparin-Sepharose (29) were prepared from strains
BWG1-7A grown in minimal medium (Dmin) to an
A600 of 1.0 at 30 °C. A 74-bp fragment ( Footprinting Analysis--
The footprint assay mixture (final
volume, 50 µl) contained 2-3 ng (~20,000-30,000 cpm) of
32P end-labeled DNA fragment, 5 µg of poly(dI-dC), and
15-30 µg of protein extract in the footprint buffer described above.
Bovine serum albumin (10 µg/µl) was added to give the same amount
of protein in each reaction. The DNA fragment used was prepared by isolating the EcoRI-XhoI GCV2 fragment
( GCV2 Is Induced by Glycine and Methionine and Repressed in Rich
Medium by Nitrogen Sources--
Previous work has shown that the
GCV2 gene encoding the P component of the GDC is regulated
by excess glycine in the external medium, that this effect is
restricted almost solely to glycine (8), and that similar responses
have been demonstrated for expression of GCV1 and
GCV3 (7, 9). We have determined whether the LPD1
gene encoding the fourth component of the glycine decarboxylase complex
is regulated by glycine. LPD1 showed a fairly high level of
expression in cells grown in minimal medium, and no significant effect
on expression with the addition of glycine (Fig.
1A). This is not surprising
because in yeast LPD1 encodes lipoamide dehydrogenase, which
is a subunit for at least three other multienzyme complexes including
pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and
the branched chain 2-oxoacid dehydrogenase (11, 12). Fig. 1A
illustrates the extent of the glycine control over GCV1,
GCV2, GCV3, and LPD1 expression in the
same strain.
This paper is primarily concerned with the glycine control system;
however, given the role of the GCV genes in one-carbon metabolism and because glycine can act as a nitrogen source, we also
investigated whether any other products of one-carbon metabolism or
nitrogen sources affected control of GCV2. Fig.
1B illustrates the levels of expression seen when cells are
grown in Dmin alone, Dmin supplemented with one of the one-carbon
metabolites (L-methionine), rich glucose medium (YPD), and
Dmin medium in which glycine, L-proline, L-glutamine, or L-asparagine replaced
NH4+ as the nitrogen source (GLYmin,
PROmin, GLNmin, and ASNmin, respectively) or a mixture of good nitrogen
sources (GLN + ASN + NH4+)min. These
results show that the GCV2 gene can be regulated over an
150-fold range, of which about 20-fold is due to repression in rich
medium and 7-fold due to glycine induction. No significant change was
observed for the addition of separate components of the one-carbon
metabolites to Dmin except for L-methionine, which caused
an approximate 2-fold induction of GCV2. Repression in rich
medium is probably due to a form of nitrogen-catabolite repression because growth of cells on L-glutamine as nitrogen source
led to a 3-fold decrease in GCV2 expression and on
L-proline a 50% increase relative to that seen on
NH4+ as nitrogen source. A mixture of
L-glutamine, L-asparagine, and NH4+ repressed GCV2 to the
level seen in YPD (Fig. 1B). Therefore, GCV2
expression is induced by glycine and L-methionine and is repressed by rich nitrogen substrates.
Localization of Control Elements in the GCV2 Gene--
Important
control regions were located by deletion analysis of the upstream
region of the GCV2 gene. Plasmid pRH2 contains 1.37 kilobase
pairs of GCV2 sequence fused to the E. coli lacZ reporter, including 1 kilobase pair of GCV2 upstream
sequence. This construct when integrated as a single copy at the
URA3 locus retained all of the known control elements.
Unidirectional deletion analysis (Fig.
2A) indicated that the glycine
response was mediated by sequences between
Repression in the absence of glycine was partially relieved (2-fold) on
deletion of sequences to
To further dissect the glycine-specific control region, overlapping
fragments between
The constructs shown in Figs. 2 and 3 were also used to locate the
L-methionine response region of the GCV2 gene
(Fig. 1B). The 11-bp sequence that was important for the
glycine response was also involved in the L-methionine
response, and the same sequences are responsible for the regulation of
GCV2 when cells are grown in glycine- or
L-methionine-supplemented medium. A GCV1-lacZ
fusion construct was also found to respond to L-methionine,
whereas a GCV3-lacZ construct did not (data not shown).
To further delimit the important bases in the 11-bp sequence that
partly restored the glycine response, the region was subject to
site-directed mutagenesis. The mutations made included one that altered
4 bp in the GCN4B core 5'-TGACTC-3' consensus to 5'-GGTACC-3' and
another that altered the 5'-CTTCTT-3' hexanucleotide sequence to
5'-GGTACC-3' (Fig. 2A; RH8 and RH9). Mutation of the GCN4B
site made a small difference to the level of basal expression; however,
the change to the 5'-CTTCTT-3' sequence led to over 2-fold increase in
the basal level expression, reflecting a partial loss of glycine
repression. Because there were also effects on basal GCV2
expression from sequences between
The above results indicate that sequences between
The main deletion analysis (Fig. 2A) also revealed control
elements regulating GCV2 expression under other conditions.
In YPD medium, the gene is repressed over 20-fold relative to
expression on minimal medium (Fig. 1B). This repression
occurred in deletions down to
The above results have localized sequences essential for repression of
GCV2 in rich medium including nitrogen catabolite regulation (up to 20-fold). For the glycine response, an essential
"core" 11-bp region has been identified, although flanking
upstream sequence(s) also appear to play a role. Because the glycine
response is common to GCV1, GCV2, and
GCV3 genes and very little is known about the molecular
mechanisms regulating expression of genes for one-carbon metabolism in
S. cerevisiae, we have concentrated on this aspect of the regulation.
The Glycine Regulatory Region from GCV2 Mediates Repression When
Inserted in a Heterologous CYC1 Promoter, and This Repression Is
Relieved by Glycine and L-Methionine--
To test whether
the 11-bp essential region and adjacent sequences involved in glycine
induction have activity in a different sequence context, the 42-bp
fragment (
These results confirmed that the 42-bp glycine regulatory region (GRR)
of GCV2 from The GCV1 Gene Is Regulated Positively by a Very Similar Element to
the GRR That Regulates the GCV2 Gene Negatively--
Deletion analysis
of the GCV1 upstream sequence gave the interesting result
that unlike the negative control seen for GCV2, deletion of
sequences (between
These results indicate that the GCV1 GRR element may
function as either an activator or a repressor depending on its
context, but the response to glycine is retained. This has interesting implications for the way that this cis-acting element functions.
Protein Specifically Binds to the GCV2 Sequence (
Two fragments were generated by cutting the
This complex could be effectively competed by the unlabeled DNA
sequence from
There was no difference in the DNA-protein complexes formed using
extracts prepared from cells grown on inducing concentrations of
glycine and those from cells grown in the absence of glycine (data not
shown). This may indicate that the putative transcription factor is
present constitutively and is activated by modification or ligand
binding in the presence of excess glycine or methionine in the medium.
The availability of an in vitro binding assay enabled screening for possible interactions between the GRR-binding protein and
low molecular mass metabolites related to one-carbon metabolism.
Tetrahydrofolate Affects the Binding of the Putative Glycine
Response Protein to the Control Region of Both the GCV2 and GCV1
Genes--
Complicated signal transduction pathways often mediate
activation or repression of a transcription factor in response to a stimulus. For a few systems, however, low molecular mass metabolites can act as ligands to activate a transcription factor (e.g.
the Hap1p, heme-activated protein; Ref. 31). We therefore tested products or intermediates of one-carbon metabolism, including glycine,
L-methionine, folic acid, and H4folate. Of the
compounds tested, H4folate had a marked effect on the
binding of the protein in the gel mobility shift assays.
Complex formation responded at concentrations of H4folate
between 10 and 50 µM and was responsive 3-fold in the
range up to 1 mM (Fig.
8B). Control experiments
adding the same concentrations of buffer and 2-mercaptoethanol needed
to stabilize H4folate did not affect formation of the
complex. Other compounds tested, including folic acid, folinic acid,
glycine, and L-methionine had no effect at concentrations
up to 10 mM, which were beyond physiological levels.
Because the GCV1 gene is also regulated by glycine, we repeated the above experiments with a 31-bp fragment ( Limitation of Folate Synthesis Affects the Glycine Response of GCV2
in Vivo--
To test whether the above in vitro results
with H4folate binding were relevant to the control of
one-carbon metabolism in vivo, we tested the effect of
inhibiting H4folate biosynthesis on glycine induction of
GCV2. For this we used the recently generated fol1 mutant (YUG1) provided by Dr. Johannes H. Hegemann
(Heinrich-Heine University, Düsseldorf, Germany). This strain
requires folinic acid for growth. By growing the mutant at different
extracellular concentration of folinic acid (up to 250 µg/ml), it was
found that the strain required 50 µg/ml for growth to occur, and at this level the growth rate was reduced to 7.4 h (form 2.3 h
at 100 µg/ml), indicating that the folate pool was limiting growth.
Cells of YUG1 transformed with the GCV2::lacZ
construct were grown to late exponential phase
(A600 of 1.3) in Dmin containing 50 µg/ml of
folinic acid and then transferred to fresh medium in the presence and
absence of glycine (10 mM) and folinic acid (100 µg/ml),
and the level of induction of GCV2 was determined by
measuring Previous studies of the expression of GCV1, GCV2, and
GCV3 have shown them to be regulated in response to the
presence of glycine in the external medium (7-9). We have here shown
that GCV2 can be regulated sensitively over a broad (about
150-fold) range from maximal induction in cells growing in minimal
medium with glycine as the sole nitrogen source to maximum repression in rich medium. The glycine decarboxylase complex thus appears to be
controlled to balance the requirement for one-carbon metabolites versus the need for nitrogen.
In addition to the glycine response we have identified a control
capable of modulating expression up to 20-fold in response to growth on
different nitrogen sources or on rich medium. Most of this effect is
localized to a 23-bp region ( Deletion analysis has indicated that glycine control is a response to
the binding of a protein at a GRR. Site-directed mutagenesis localized
the main effects to a CTTCTT element within GRR. However, it is also
clear that there is a requirement for additional upstream sequences for
full wild-type response to glycine of GCV2. These sequences
cannot on their own confer any response on the gene and are not
absolutely essential but serve to modulate the degree of expression
from the adjacent CTTCTT element. It appears therefore that a glycine
response protein transcription factor binds to the 6-bp motif, and this
binding is stabilized by flanking sequences. The footprinting data are
consistent with the genetic analysis because they indicate the
existence of a protein that protects the 22-bp sequence from One of the significant and interesting findings of the work is that
tetrahydrofolate could affect the affinity of in vitro binding of the protein in the gel mobility shift assay when using the
GRR from GCV1 or GCV2 and that folate limitation
affects the in vivo glycine response of GCV2.
This provides some insight into the mechanism used by the cell to
detect excess glycine and signal this to the transcription apparatus.
Because tetrahydrofolate is the molecule to which the one-carbon
derivatives in their various oxidation states are substituted, the
control appears to be mediated directly at the level of this compound
or one of its derivatives rather than through a signal transduction
pathway. We have tested in vitro a range of compounds
including glycine analogues, but only tetrahydrofolate has been found
to function at a reasonable concentration. In cells the concentration
of total reduced folates is reported to be between 10 and 40 µM (33, 34). In our experiments the extent of formation
of the complex was affected by concentrations as low as 10 µM with the changes being sigmoidal in the range from 10 to 500 µM. Although this appears to be above in
vivo levels, many proteins that bind tetrahydrofolates in
vivo have been found to show a greater affinity for the
polyglutamylated species, and the binding affinity decreases as the
extent of ( Clearly the use of tetrahydrofolate or one of its derivatives as a
signal of one-carbon status is very appropriate. How does this get
translated into activation of the glycine decarboxylase complex genes?
The glycine decarboxylase complex is activated to some extent in
minimal medium, and the cell would be forming serine via the glycolytic
pathway and converting it to glycine via the complex. The concentration
of tetrahydrofolate may therefore be at an intermediate level (compared
with growth in rich medium or with growth in minimal medium with
glycine) because the reaction would be limited by the cellular pool
level of serine. In the presence of excess glycine (when the glycine
response is observed), the cell would be able to spare serine and to
obtain nitrogen from the glycine in the absence of more readily
assimilated nitrogen sources. The excess glycine in the cytoplasm of
the cell would inhibit the serine-to-glycine conversion via serine
hydroxymethyltransferase, and if so this would alter the generation of
5,10-CH2-H4folate to lead to higher levels of
the uncharged H4folate. This may then signal an increase in
the activity of the mitochondrial glycine decarboxylase to generate
serine from glycine and therefore also increase the flux of one-carbon
metabolites. Because there are three principal donors to the one-carbon
pool in yeast (serine, glycine, and formate), a similar control via
H4folates may also be found over the relevant genes.
Because H4folate increases the binding to DNA of protein in
the gel mobility shift experiment and because the DNA motif in one gene
(GCV1) can act as an activator and in the other
(GCV2) can act as a repressor, it is difficult to predict
how tetrahydrofolate binding alters the transcriptional apparatus until
the gene for the DNA-binding protein can be identified. It is a
continuing aim of this work to determine what other genes involved in
one-carbon metabolism are similarly regulated and to identify the genes
encoding the DNA-binding proteins that mediate the control at the level of transcription.
The GRR of GCV genes, 5'-CATCN7CTTCTT-3' is also
found in the promoter of the DFR1 gene encoding
dihydrofolate reductase (at
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
351-378 bp of
GCV2) and pRH2 (1.37-kilobase pair
KpnI/XbaI GCV2 fragment). Plasmid pRH3
contains a 585-bp EcoRI/XbaI GCV2
fragment 9
206 to 378 bp) isolated from pGSD2.5
5. Each
GCV2 fragment was fused in-frame upstream of the
lacZ gene in YIp358 (23).
312
and
291 and the CTTCTT motif of GCV2 were mutated to
KpnI restriction sites as described (25), and the PCR
products were cloned into YIp358. Constructs with the GCN4 mutated at
312 and
291 were designated pRH7 and pRH8, respectively, and the CTTCTT mutated construct was pRH9. Further deletions were constructed from pRH7 and pRH8 by cloning the 690- and 669-bp
KpnI/XbaI fragments into YIp358 to produce pRH10
and pRH11, respectively. To localize the glycine regulatory element,
five sets of double-stranded oligonucleotides with EcoRI
overhangs were prepared from pairs of annealed synthetic oligonucleotides for ligation into pRH4 upstream of the 5' end of the
truncated GCV2 gene. The resulting constructs were sequenced to determine the orientation of each insertion.
351 to
313 of GCV2 and pRH8 containing
351 to
289 of
GCV2 were isolated after cutting with XbaI.
Subsequent blunt-end ligation created pRH12 and pRH14. pRH9 was used to
make another window deletion construct (pRH13) by cutting with
KpnI and then religating the larger fragment. This deleted
the sequence between two potential Gcn4p binding sites (
310 and
289) of GCV2.
194 to
157 harbored the
GCV1 glycine regulatory region as determined by broad
deletion and computer analysis.
410 to +1328 (relative to the
start codon) was amplified from genomic DNA by PCR and the
BamHI fragment of GCV3 (
371 to +1291) was
cloned into pTZ19. Subsequently, the GCV3 BamHI to
SphI (
371 to +53) fragment was subcloned into YIp356 (23)
and fused in frame to the lacZ reporter gene.
1026 to +96 (SphI-KpnI) fragment of
GCV1 was cloned into YIp357R (23) and fused to
lacZ in this study. Deletion constructs were also made from
HindIII-KpnI (
310 to +96 of GCV1) and XhoI-KpnI (
130 to +96) fragments by
cloning into the same vector. The LPD1-lacZ fusion
reporter construct (pDS1) has been described previously (8).
322 to
248 (relative to start codon)
was amplified by PCR using primers harboring XhoI
restriction sites and the internal 42-bp XhoI fragment was
cloned into the XhoI site within the CYC1
promoter region of pLG
-312S (26). The GCV1 fragment
193
to
162 with XhoI cohesive ends was produced by annealing
appropriate oligonucleotides and after phosphorylation was cloned into
pLG
-312S or pLG
-312SS, which is identical to pLG
-312S except
that the UAS sites in the promoter region of the CYC1 gene
were removed by cutting with SmaI and SalI, and religating after filling in the SalI site.
-Galatosidase Assay--
-Galatosidase assays were
performed as described previously (8). Yeast strain BWG1-7A was either
grown to an A600 of 0.5 (for nitrogen source
regulation) or to an A600 of 1.0 and then transferred to fresh medium and incubated a further 2 h before harvest (for glycine and L-methionine response analysis).
Cells of strain YUG1 were grown to an A600 of
1.3 and then transferred to fresh medium after centrifugation and
washing and incubated for appropriate time before harvest.
322
to
248) of GCV2 was amplified by PCR and was cut with
appropriate restriction enzymes. Termini were filled using the Klenow
fragment of DNA polymerase in the presence of
[
-32P]dATP/dCTP, and the fragment was isolated by
polyacrylamide gel electrophoresis. 8 fmol of the labeled DNA was added
if not specified otherwise, and 2 µg of poly (dI-dC) was added to
prevent nonspecific binding of proteins to the DNA. Reaction mixtures
containing footprint buffer (27) were incubated at room temperature for
20 min before loading on a 5% (w/v) polyacrylamide gel and separated
by electrophoresis at 7.5 V cm
1 for 2 h. Gels were
dried, and radioactivity was scanned by a Phosphoimager (Bio-Rad) to
quantify the amount of the retarded species as a DNA-protein complex in
comparison with the free DNA. Densitometric scanning was performed for
the H4folate effect analysis by Imaging Densitometer
(Bio-Rad).
351 to
148) from pRH1 and then cutting this fragment with
AluI generating an EcoRI-AluI fragment
(
351 to
218). The 5' EcoRI terminus was end-labeled with
[
-32P]dATP, and the fragment was isolated by
polyacrylamide gel electrophoresis. After a 20-min incubation at room
temperature, freshly diluted DNaseI (4.5 units) was added, and the DNA
digestion was proceeded for 1 min at room temperature. The reaction was
stopped by adding 140 µl of stop solution containing 192 mM sodium acetate, 32 mM EDTA, 0.14% SDS, and
64 µg/ml yeast RNA. DNA fragments were extracted once with
phenol/chloroform (1:1) and precipitated with ethanol and 0.3 M sodium acetate. The pellet was rinsed with 95% ethanol and resuspended in 4 µl of loading dye (95% v/v formamide, 10 mM EDTA, 1% bromphenol blue, 1% xylene cyanol). The DNA
fragments were denatured for 1 min at 90 °C prior to electrophoresis
on an 8% sequencing gel. The G + A sequencing was prepared from the same fragment (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, the response to glycine of expression
of the four genes encoding the GDC components. Upstream sequences of
the GCV1, GCV2, GCV3, and
LPD1 genes were fused in-frame with the lacZ
reporter gene, and each was transformed separately into strain
BWG1-7A. -Galactosidase assays were performed in triplicate.
B, expression of the GCV2 gene in cells grown on
different media. Cells containing the full-length GCV2-lacZ
reporter construct integrated in strain BWG1-7A as a single copy were
grown to an A600 of 0.4-0.5 in the different
media indicated and harvested for
-galactosidase assay. Expression
in minimal medium (Dmin) and Dmin supplemented with 2 mM
L-methionine and also the effect of different nitrogen
sources compared with complex medium (YPD) on GCV2
expression are shown.
-Galactosidase assays were performed in
triplicate. Error bars represent the standard
deviation.
313 and
267, with
complete loss of the glycine effect on deletion to
267. Glycine
induction is actually due to a loss of repression of GCV2
because deletion led to a constitutively higher level of gene
expression as is clearly seen in Fig. 2B, which shows the
response of the deleted constructs to different concentrations of
external glycine.
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Fig. 2.
A, deletion analysis and site-directed
mutagenesis of the GCV2 promoter to localize sequences involved in the
glycine response. Constructs were made as described under
"Experimental Procedures" and integrated in strain
BWG1-7A as single copies. Fold induction of expression
from Dmin ( gly) to Dmin supplemented with 10 mM glycine (+gly) is indicated in
parentheses. The extent of the deletions is indicated on the
left of the diagram with the locations of potential GCN4
sites, the GATA element, and the CTTCTT motif as indicated. The
single line in the window deletion constructs (pRH12 to 14)
represents sequences deleted from pRH1. The crosses
represent mutated motifs for the putative GCN4 and CTTCTT elements.
Cells were grown and assayed in triplicate for
-galactosidase as
described in the legend to Fig. 1A, and the data are given
with the standard deviation of the mean. B, the glycine
response element acts as a repressor in the GCV2 gene. The
glycine response of four representative deletion constructs outlined
above was determined at different concentrations of glycine in the
external medium. Open squares represent the full-length
construct in pRH1, closed squares represent pRH2, open
circles represent pRH3, and closed circles represent
pRH4.
-Galactosidase assays were performed in triplicate; all errors
were less than 25%.
289. This partial derepression was also
observed in the window deletion construct pRH13 (Fig. 2A)
with deletion of sequences between
310 to
289. Window deletions from
313 to
267 and from
289 to
267 showed complete loss of repression. Therefore, sequences between
289 and
267 are absolutely required for glycine-specific repression of GCV2 expression
and an additional important sequence between
310 and
289 affects the level of this control because its deletion led to a consistent 2-fold increase in basal expression on Dmin without loss of glycine control.
291 and
266 were inserted in both forward and
reverse orientation at the 5' end of the truncated GCV2-lacZ
in pRH4 (the largest promoter lacking glycine-specific control). Among
these constructs, those with the 11-bp fragment (5'-TGACTCTTCTT-3')
inserted in either forward or reverse orientation restored the glycine
response of GCV2 (Fig.
3A). However, this fragment
did not completely repress GCV2 expression, which is consistent with the window deletion data above, because it only showed
a 2-fold effect compared with the wild-type (pRH1), which showed about
4-fold repression. This fragment contains the motif 5'-TGACTC-3', which
is a potential binding site for Gcn4p, Yap1p, and Bas1p transcription
factors, as well as an additional 5'-CTTCTT-3' motif. This 11-bp
sequence was sought in the other known glycine responsive genes,
GCV1 and GCV3 (7, 9). The promoter of GCV1 shares remarkably strong homology with GCV2
over about 50 bp including the 5'-CTTCTT-3' motif (at
177; Fig.
3B). For GCV3 there is less homology with either
GCV1 or GCV2 in the promoter region, although
there is a region around a CTTCTT motif (at
262) that shows a fair
degree of sequence conservation across all three genes.
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Fig. 3.
Sequences of GCV2 (between
291 and
266 relative to the start codon) that can restore the
glycine response to the truncated GCV2 promoter in
pRH4. A, the fragments from 1 to 5 indicated in the
diagram were cloned in both forward and reverse orientations at the 5'
boundary of the GCV2 sequence in pRH4. The resulting
constructs were integrated as single copies in strain BWG1-7A and
assayed for
-galactosidase in triplicate as described in the legend
to Fig. 1A; all errors were less than 26%. B,
comparison of the promoter regions of the glycine-responsive genes
(GCV1,
206to
132; GCV2,
315 to
240;
GCV3,
314 to
241). The asterisks indicate
identity to the central GCV2 sequence of either
GCV1 or GCV3.
313 to
289 (just upstream of the
11-bp sequence) and this contains another potential GCN4 site, this
(GCN4A; at
312) was also mutated to 5'-GGTACC-3'. There was no effect
of this change on the basal level or glycine responsiveness of
GCV2 expression (Fig. 2A; RH7).
313 and
267
affect the overall response to glycine (and L-methionine) and that an 11-bp fragment 5'-TGACTCTTCTT-3' can confer some glycine response with the 5'-CTTCTT-3' at
286 playing an important part in
this glycine effect. There are, however, additional sequences within
the
313 to
289 region that are involved in setting the level of
basal expression to accomplish the full response.
227 but was lost on further deletion to
205 (data not shown). This response to growth on rich medium is
therefore separate from that to one-carbon metabolites such as glycine
and L-methionine. The nitrogen source repression seen in
L-glutamine-containing minimal medium was localized to the
same region as that for the rich medium response using the deletion
constructs (Fig. 2A).
309 to
267) spanning the important core and flanking
sequences was inserted into the XhoI site located between
the UASC and TATA box elements of a complete CYC1 promoter fused to a lacZ reporter (Fig.
4). Two constructs were tested; one
contained one 42-bp fragment, the other carried two inserts in a tandem
duplication. In this relatively strong promoter, insertion of one
element led to a 2-fold repression of expression, and much of this
repression was relieved on adding glycine to the medium (Fig. 4). When
two 42-bp fragments were inserted, repression was greater than 4-fold,
and the addition of glycine caused a 3-fold "induction" to 75% of
the level seen in the intact CYC1 promoter. Similar results
were obtained on addition of L-methionine instead of
glycine as an inducer (data not shown). These results demonstrate
clearly that the elements confer a glycine response, and this is true
even if changes in promoter spacing were affecting expression.
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Fig. 4.
The 42-bp glycine regulatory region from
GCV2 confers repression on an heterologous promoter
that can be relieved by glycine. A, the arrangement of
elements in intact UASC-containing plasmid pLG -312S
indicating the XhoI sites used for insertion of the glycine
response element. B, the GCV2 region from
309
to
267 was inserted as either one copy (forward orientation) or two
tandem copies (reverse orientation) at the XhoI site of the
CYC1-lacZ promoter of pLG
-312S. Each construct was
integrated in strain BWG1-7A and assayed for
-galactosidase in
triplicate.
309 to
267 is capable of causing repression of gene expression in an heterologous system with a more highly expressed promoter than that of GCV2 and that it carries
element(s) needed for glycine and L-methionine to relieve
this repression as is seen in the native GCV2 gene.
206 to
130) led to a loss of glycine induction
(data not shown). Because the GCV1 sequence can act as an
activator,
193 to
162 of GCV1 was inserted into the
blind (UAS-less) CYC1-lacZ reporter plasmid pLG
-312SS at the XhoI site with results that were virtually the opposite
of those seen for the homologous GCV2 element (Fig.
5A). One copy caused strong
activation even in the absence of glycine in the medium, and addition
of glycine caused a modest induction. When four copies of the construct
were present in tandem array, there was a very substantial activation
of the reporter in the absence of glycine and a greater than 10-fold
further induction on the addition of glycine. The homologous elements
from GCV1 and GCV2 therefore both introduce
glycine responsiveness but differ in whether they act to repress or
promote transcription, reflecting the situation in the native genes.
Because this region contained the sequence homologous to the functional
42-bp element in GCV2 (Fig. 3B), the region of
strong homology (
193 to
162) of GCV1 was also inserted
into the XhoI site of the CYC1-lacZ reporter plasmid pLG
-312S. In this context (Fig. 5B) the sequence
now more closely resembled the situation seen with GCV2,
because it acted as a repressor with a greater effect of more copies,
still retaining glycine responsiveness. The effect of context was
further highlighted by inserting the GCV1 glycine regulatory
region (
193 to
162) in front of the truncated GCV2
promoter in pRH4. This restored a glycine response to the
GCV2 construct, and in this context the GCV1
element was acting in a similar way to the GCV2 element
(Fig. 2A; RH15).
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Fig. 5.
The 31-bp glycine regulatory region from
GCV1 confers a glycine response on UAS-less and
UASC-containing heterologous
CYC1-lacZ promoters. The element
activates or represses transcription according to con- text.
A, the GCV1 region from 193 to
162 was
integrated as either one copy in forward (F) or reverse
(R) orientation or four copies (reverse orientation) at the
XhoI site of the blind CYC1-lacZ promoter in
pLG
-312SS. B, the same GCV1 sequence was
inserted as either one or two copies (in both forward (F)
and reverse (R) orientations) into the CYC1-lacZ
promoter of pLG
-312S containing an intact UASC. Each
construct was transformed in strain BWG1-7A and assayed for
-galactosidase in triplicate. Specific activity of the
constructs was determined as described under "Experimental
Procedures." Error bars represent the standard
deviation.
309 to
267)
Responsible for Glycine Control--
To determine whether any protein
bound to the GRR of GCV2, gel mobility shift analysis was
performed initially using a DNA fragment spanning the region of
GCV2 from
322 to
248. Nuclear extracts were obtained
from cells grown in minimal medium with or without glycine. The DNA
sequence from
322 to
248 was amplified by PCR and in a mobility
shift assay was found to form one major complex with the nuclear
protein extract. Similar complex formation was observed using the
internal 42-bp XhoI fragment (
309 to
267; Fig.
6A). This complex was not
formed in assays using cell extracts treated with proteinase K (data
not shown).
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Fig. 6.
Gel mobility shift analysis of protein
binding to the glycine regulatory region of GCV2.
Nuclear extracts preparation was performed as described under
"Experimental Procedures," and the GCV2 sequences used
are indicated in the diagram. A, protein binding to the
332 to
248,
309 to
267 (XhoI),
322 to
295
(MboII), and
295 to
248 (MboII) fragments.
The first lane in each set is a no protein control. 8 fmol of labeled
DNA and 5 µg of protein were added in other reaction mixtures.
Arrows indicate the major DNA-protein complex. B,
effect of mutation of the CTTCTT sequence (
286) to GGTACC on binding
of the XhoI fragment. The titration of total protein
concentration for wild-type and mutant sequences is shown.
C, competition using unlabeled wild-type (W.T. or
w.t.) and mutant sequences. 5 µg of protein extract was
used in every lane.
322 to
248 fragment at
an MboII site (cuts at
295). No protein bound to the smaller fragment (Fig. 6A), and although binding occurred to
the larger one, it was not as extensive as that to the longer
XhoI fragment (
309 to
267). This indicated that although
the protein could bind between
267 and
295, for strong binding
there was also a requirement for bases up to 14 bp further upstream.
This correlated with the in vivo results from the genetic
studies, which showed that although the major control element of
GCV2 was between
289 and
267, flanking sequence up to
310 were needed for the full response.
309 to
267 but not by the same sequence with a
mutation of the CTTCTT motif (Fig. 6C). Moreover the
specificity of this binding was further shown by the greatly reduced
ability of the mutant DNA sequence to bind protein in the extract (Fig. 6B). DNaseI footprinting of the protein-DNA complex (Fig.
7) showed that the region from the
5'-CATCN7CTTCTT-3' motif was protected and that binding of
the protein led to an increase in DNaseI susceptibility of the bases
immediately 3' of the footprint (asterisk in Fig. 7),
indicating an effect of protein binding on the topology of the DNA in
this region. From Fig. 3B it can be seen that the protected region corresponds to the sequence of greatest homology between GCV1, GCV2, and GCV3, with the
flanking CATC and CTTCTT motifs most conserved. These results are fully
in accord with the genetic data presented above because the binding of
the protein depends on the region containing the CTTCTT motif but is
augmented by the bases further upstream to include the CATC. The
protein is therefore an excellent candidate for a transcription factor
that mediates the glycine control.
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Fig. 7.
DNaseI footprint of protein bound to the
GCV2 glycine regulatory region. The preparation
of DNA-binding protein, binding, and the footprint reaction were
carried out as described under "Experimental
Procedures." The protein extract was incubated with a 5'
end-labeled EcoRI-AluI GCV2 fragment
( 351 to
218). The lanes 1 and 2 are free DNA
digested with DNaseI, and lanes 3-5 had increasing amounts
of protein added to the reaction (15, 23, and 30 µg, respectively).
Sequences protected from DNaseI digestion are shown in the
box. A densitometry scan of lanes 2 (gray) and 5 (black) is also shown. The
numbers indicate the base positions with respect to the
start codon. The asterisk indicates the site of enhanced
cleavage.
193 to
162)
of GCV1 encompassing the GRR region that contains the core CTTCTT motif. Similar results were obtained (Fig. 8C), which
indicated that the putative GCV1 promoter region can bind
the same proteins as GCV2 and that there is a similar effect
of H4folate on the binding.
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Fig. 8.
Effect of different concentrations of
H4folate on binding of proteins from yeast strain BWG1-7A
to the GRR of GCV2 and of GCV1.
A and B, formation of the complex with the 309
to
267 (XhoI fragment) of GCV2 at increasing
concentrations of H4folate was determined by densitometry
following gel mobility shift analysis (A). Relative optical
density was measured in quadruplicate in different experiments
(B). Error bars represent the standard deviation.
C, comparison of gel mobility shift assays from protein
extracts prepared by heparin-Sepharose chromatography eluting with an
(NH4)2SO4 gradient as described
under "Experimental Procedures." For each panel the left-hand
set of 10 lanes represents the control without added
H4folate (the first lane is no protein control);
the right-hand set is the same fractions with 1 mM H4folate added to the assay. Upper
panel was obtained using the XhoI fragment of
GCV2, and the lower panel was obtained using the
193 to
162 fragment of the GCV1 gene.
-galactosidase activity at intervals. From Table I, it can be seen that under conditions
in which folate was limiting or depleted the GCV2 gene was
not inducible by glycine but that a response was obtained in the
presence of folinic acid. The results support the hypothesis that a
folate species plays a role in the cell in the glycine response of
GCV2 and therefore in regulation of one-carbon
metabolism.
GCV2 expression (pRH1) in the fol1 strain YUG1 under different
conditions of folate limitation
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
227 to
205) that includes a 9-bp
palindromic sequence adjacent to one copy of a GATA element typically
associated with genes regulated by nitrogen catabolite repression (19).
There may be some involvement of a protein binding to the GATA site,
although this situation would be different from that seen for most
other genes for which multiple copies of these sites are required (20,
32). Adding L-methionine to the medium also led to an
elevation of GCV2 and GCV1 expression to a lesser
extent than glycine. This control is mediated through the same motifs
in the promoters as the glycine response. Although the advantages to
the cell of up-regulating the glycine metabolic genes in response to
excess glycine is obvious, those resulting from elevated expression in
response to L-methionine are less so.
L-Methionine seems metabolically rather remote from the
sensing system involved in regulating transcription because in another strain of S. cerevisiae we have seen
L-methionine work in the opposite direction to repress the
GCV2 gene. This and the nitrogen control require more
extensive investigation because here we have concentrated in much more
detail on the glycine response.
277 to
299, which includes the CTTCTT motif and an upstream flanking CATC
sequence. Similar motifs to those involved in the glycine response of
GCV2 exist in the GCV1 and GCV3 genes.
It is interesting to note that the gene with the least similarity to
the others in the glycine regulatory region of the promoter
(GCV3) showed no response to L-methionine unlike the other two genes.
glutamyl conjugation decreases (35-37). It is also not
yet clear whether it is H4folate or one of its derivatives
that is active in vivo because we have been unable to test
the full range of folate derivatives. Folic acid and folinic acid were
inactive in the assay system used.
377), which catalyzes de novo
synthesis of H4folate. The spacing between the CATC and
CTTCTT motifs seen in the GRR of GCV1 and GCV2
was also conserved in the DFR1 promoter. The importance of H4folate in the regulation of GCV genes, and the
existence of the GRR in the DFR1 may therefore give an
insight into the regulation of one-carbon metabolism in S. cerevisiae.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Andrew Bognar for the
GCV1 gene and Dr. Leonard Guarente for providing yeast
strain BWG1-7A and plasmid pLG-312S. We also thank Dr. Johannes
Hegemann for yeast strain YUG1.
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FOOTNOTES |
---|
* This work was supported by an Australian Research Council grant.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.
Supported by Australian Postgraduate Awards.
§ Present address: Dept. of Biology, Massachusetts Inst. of Technology, Cambridge, MA 02139.
¶ To whom correspondence should be addressed. Tel.: 61-2-9385-2030; Fax: 61-2-9385-1050; E-mail: i.dawes{at}unsw.edu.au.
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ABBREVIATIONS |
---|
The abbreviations used are: GDC, glycine decarboxylase multienzyme complex; 5, 10-CH2-H4folate, 5,10-methylenetetrahydrofolate; H4folate, tetrahydrofolate; GLYmin, glucose minimal medium with glycine as sole nitrogen source; GLNmin, glucose minimal medium with glutamine as sole nitrogen source; ASNmin, glucose minimal medium with asparagine as sole nitrogen source; PROmin, glucose minimal medium with proline as sole nitrogen source; Dmin, glucose minimal medium with ammonium as sole nitrogen source; GCN4, binding site of Gcn4p transcription factor; GRR, glycine regulatory region; UASc, upstream activation site controlling CYC1 gene expression; bp, base pair(s); PCR, polymerase chain reaction.
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REFERENCES |
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