(Received for publication, August 1, 1994; and in revised form, December 20, 1994)
From the
The LEU4 gene of Saccharomyces cerevisiae and
the enzyme encoded by LEU4, -isopropylmalate synthase,
occupy a special position in amino acid metabolism.
-Isopropylmalate synthase catalyzes the first committed step in
leucine biosynthesis. However, the reaction product
-isopropylmalate is not only an intermediate in the leucine
biosynthetic pathway, but also functions as co-activator of at least
six genes, both within and outside of the leucine pathway. The
metabolic importance of
-isopropylmalate appears to be reflected
in the surprisingly multifaceted regulation of LEU4 expression. This report describes an analysis of functional cis elements in the LEU4 promoter. Five such elements
were identified. Three distal elements, designated UAS
,
GCE-A, and GCE-B, are responsible for regulation by the regulatory
proteins Leu3p and Gcn4p, respectively. The incremental activation of LEU4 by these elements is additive and independent. In
addition, two proximal elements were localized. One of these conforms
to the TATA consensus sequence and exhibits high affinity for TATA
binding protein. The other element shows strong sequence identity with
the Bas2p binding site and appears to be involved in basal and
phosphate-mediated regulation of LEU4.
In the yeast Saccharomyces cerevisiae, the genes for a
particular metabolic pathway are generally unlinked and located on
different chromosomes. Each gene has its own promoter and regulatory
sequences. The promoters of these RNA polymerase II-transcribed genes
typically contain at least one TATA box that serves as focal point for
the assembly of the preinitiation complex. A minimal promoter without
upstream regulatory sequences is capable of directing basal level
transcription only. Upstream activating or repressing sequences (UASs ()or URSs), by interacting with their cognate
transcriptional regulators, can cause intricate and complex responses
to environmental
signals(1, 2, 3, 4) .
The LEU4 gene and the enzyme encoded by LEU4 (-isopropylmalate synthase EC 4.1.3.12,
[
-IPMS]) provide an interesting example of multiple
controls.
-IPMS catalyzes the first, committed step in leucine
biosynthesis. Physiological and genetic studies have shown that LEU4 expression is regulated by the availability of leucine
and by amino acid starvation through the general control of amino acid
biosynthesis(5, 6, 7, 8, 9) .
The regulation of LEU4 by leucine is indirect and operates
through
-IPM, a product of the
-IPMS catalyzed reaction. When
leucine is in short supply, diminished feedback inhibition of
-IPMS causes the
-IPM level to rise.
-IPM subsequently
interacts with the regulatory protein Leu3p, which in turn activates LEU4 expression. Leu3p is a well-studied DNA binding protein
of the 2-Zn-6-cysteine cluster type(10, 11) . It binds
to a consensus sequence (5`-GCCGGNNCCGGC-3`, designated
UAS
) that is found in the promoters of LEU1, LEU2,
LEU4, ILV2, ILV5, and GDH1(12, 13, 14, 15, 16) .
A current model postulates that Leu3p binds to the UAS
elements regardless of whether
-IPM is present or absent.
When
-IPM is absent, Leu3p is inert or acts as a repressor of
transcription; incoming
-IPM then changes Leu3p from an inactive
(repressive) to an active
configuration(16, 17, 18) .
The
pleiotropic mode of action of the Leu3p-IPM complex and the
function of
-IPM as a more general metabolic signal (13) place LEU4 and its gene product at the hub of a
regulatory network. It was therefore important to understand in greater
detail how the synthesis of
-IPM is regulated. Specifically, we
wanted to identify functional cis elements of the LEU4 promoter and learn in what way and to what extent they contribute
to the expression of LEU4.
Here we report that the major
regulatory elements of the LEU4 promoter are a Leu3p-binding
element (UAS) and two general control response (Gcn4p
binding) elements (GCEs). The Gcn4 protein has long been known to bind
to regulatory sequences of the 5`-TGACTC-3` type and to activate
transcription of at least 30 separate genes in response to starvation
for any one of several amino acids(19) . Activation through the
UAS
and GC elements is additive and independent. In
addition, LEU4 is subject to basal level regulation that
includes a response to the phosphate concentration in the medium and is
probably mediated by the Bas2 protein. Bas2p, together with Bas1p, has
been shown to be required for basal level transcription of the HIS4 gene and to be involved in the regulation of purine and phosphate
metabolism in
yeast(20, 21, 22, 23) . Finally, the LEU4 promoter is shown to contain one functional TATA element.
Figure 1: General structure of plasmids pYH1-pYH20. The vertical arrow indicates the position of wild type and mutant LEU4 promoters. The curved arrows indicate the direction of transcription. See ``Materials and Methods'' and Fig. 2for details of construction and location of mutations.
Figure 2:
Deletions and point mutations of the LEU4 promoter and their effects on LEU4-lacZ expression. Large deletions (A) and point mutations or
small deletions (B) were generated and sequenced as described
under ``Materials and Methods'' (see also Table 1). The
5` end points of the large deletions are shown relative to the
beginning of the open reading frame of LEU4 (designated +1, see (7) ). L, UAS (dyad symmetrical center at -445); A, GCE-A
(centered on position -419); B, GCE-B (centered on
position -356); C, GCE-C (centered on position
-319; D, GCE-D (centered on position -99). The
-galactosidase activities were measured in plasmid-bearing strains
XK147-2C, XK154-8A, XK154-2D, and XK154-5B (the
pertinent genotypes are shown in parentheses) grown either in
branched-chain amino acid surfeit medium (SD plus 2 mM leucine
and 1 mM each of isoleucine and valine, SURF) or in starvation medium (SD plus 0.2 mM leucine, STARV). The numbers represent averages
of at least two independent trials with errors <20%. Filled and open circles, wild type and mutant UAS
; filled and open rectangles, wild type and mutant GCE
sequences.
Fig. 2A shows the
results of a serial deletion experiment. Plasmids containing either the
wild type LEU4 promoter (to position -679) or truncated
promoters were used to transform four different types of cells: those
that were wild type with respect to LEU3 and GCN4,
those that lacked LEU3, those that were deficient in GCN4, and those that were deficient in both LEU3 and GCN4. When cells were starved for leucine, a condition that
stimulates both LEU3- and GCN4-dependent
transcription, the full-length promoter (plasmid pYH1) supported a high
level of expression of the LEU4-lacZ fusion (specific
-galactosidase activity of 199) when LEU3 and GCN4 were both intact. When either LEU3 or GCN4 were
dysfunctional, the level of expression dropped to about 40-50% of
the high level. The level of expression dropped to very low values when
both genes were dysfunctional. These results suggested that
Leu3p-mediated control and general control of amino acid biosynthesis
are the major mechanisms by which LEU4 is regulated and that
under starvation conditions each control contributes about equally to
the final level of expression. The first deletion, extending to
position -382 and represented by plasmid pYH2, caused the LEU4 promoter to lose its response to Leu3p; the general
control response was retained, with a starvation/surfeit ratio of
greater than 3. The next two deletions, extending to positions
-332 and -315, respectively, (plasmids pYH3 and pYH4)
caused the loss of both Leu3p-mediated and general control and resulted
in a low level of expression (``basal level I,'' 17-20
units of activity) with a starvation/surfeit ratio of close to 1.
Either deletion apparently removed elements responsible for both major
controls. A deletion extending to -208 (plasmid pYH5) lowered the LEU4-lacZ expression to ``basal level II''
(8-9 units of activity), again with a starvation/surfeit ratio of
about 1. Finally, a deletion extending to -109 (plasmid pYH6)
resulted in near zero expression of the LEU4-lacZ fusion gene.
These latter results suggested the presence of two separate elements
controlling the basal expression of LEU4.
To find out
whether the Leu3p-mediated control and the general amino acid control
are the only activation mechanisms of LEU4, and to define the
relative role of the Leu3p- and Gcn4p-mediated activation more clearly,
presumptive control elements were destroyed individually and in various
combinations by creating base pair substitutions or small deletions
within the consensus sequences (Fig. 2B). The elements
chosen for mutation were the UAS element and four
segments that conform to the conserved 6 bp core sequence
(5`-TGACTC-3`) of the Gcn4p recognition
element(19, 28) . These four segments were designated
GCE-A, B, C, and D. Incapacitating the presumed UAS
sequence by creating a 9 bp deletion (-450 to -442;
plasmid pYH7) had the same effect as deleting the LEU3 gene
(compare pYH7 with pYH1 in a leu3-
2 GCN4
background), indicating that the
sequence around position -445 is indeed the only functional
UAS
of the LEU4 promoter. To avoid interference
from Leu3p regulation, the analysis of the relative importance of the
presumptive GCE boxes was performed in a UAS
-negative
background. Disabling GCE-A (plasmid pYH8) resulted in a reduction of
the starvation/surfeit ratio from 4.7, seen with plasmid pYH7, to 2.7.
A similar starvation/surfeit ratio (2.9) was obtained upon inactivation
of GCE-B (plasmid pYH9), although in this case lower absolute levels of
-galactosidase were observed. Mutating GCE boxes C and D, either
separately or together, did not significantly affect the expression of LEU4 (compare plasmids pYH10, 11, and 15 with pYH7). Mutating
both GCE-A and -B, on the other hand, led to low expression (pYH12)
that was indistinguishable from the level of expression seen with
plasmid pYH7 in a gcn4
background.
Additional permutations confirmed the above results. Thus, simultaneous
mutation of GCE-A and -C or of GCE-A and -D had the same effect as
mutating GCE-A alone (compare plasmids pYH13 and pYH14 with pYH8), and
mutation of GCE-B superimposed upon the -382 deletion (plasmid
pYH16) yielded values similar to those of plasmid pYH12. We conclude
that, of the four Gcn4p recognition elements, only GCE-A and -B are
functional. Destruction of these cis elements by site-directed
mutagenesis has the same effect as genetic elimination of the trans-acting factor Gcn4.
Figure 3:
Additive activation of LEU4-lacZ expression from UAS and GCE sequences. Plasmids
pYH1, pYH7, pYH17, and pYH12 were introduced into strain XK53-31
and
-galactosidase activities were measured as described under
``Materials and Methods'' in cells grown either under
starvation (solid blocks) or surfeit conditions (hatched
blocks). See legend to Fig. 2for definition of starvation
and surfeit. The numbers above the solid blocks indicate the -fold increase of
-galactosidase activity under
starvation conditions. UAS
,
intact UAS
element; UAS
, mutated UAS
element; GCE
, all GCE sequences are
intact; GCE
, GCE-A and -B sequences are
mutated (see Table 1), GCE-C and -D sequences are
intact.
Figure 4: DNase I footprint analysis of the proximal region of the LEU4 promoter. A, different concentrations of yTBP, as indicated across the top, were incubated with end-labeled DNA fragments of the LEU4 promoter (positions -343 to -43). DNase I footprinting was performed as described under ``Materials and Methods.'' The numbers on the left of each panel refer to positions of the LEU4 promoter relative to the start of the open reading frame (+1). They were assigned with the aid of a sequencing ladder. B, the protected regions on either strand (coding strand on top) are underlined. Potential additional protection is shown by small capital letters.
In this study, we have identified five functional cis elements that govern the expression of the LEU4 gene. The
three distal elements are are located between positions -455 and
-353 (relative to the start of the LEU4 open reading
frame) and encompass one Leu3p-binding element (UAS) and
two Gcn4-binding elements (GCE-A and -B). These three elements are
responsible for the two major controls of LEU4 that had
previously been postulated to occur on the basis of physiological
studies and genetic manipulation of trans-acting
factors(5, 9) . The two proximal elements are centered
on position -260 and on position -143, respectively. The first of
these clearly shows the properties of a functional TATA box: it is
protected by and strongly interacts with a TATA-binding protein, and
its destruction causes a drastic decrease of LEU4 expression.
This TATA box is unusually far removed from the first major
transcription start site (about 195 versus 40-120 bp for
most yeast promoters). The element centered on position -143
possesses the features of a Bas2p-binding site(20) . Expression
from this site apparently proceeds without an additional TATA element.
This is not uncommon; TATA-independent transcription from a Bas2p site
was previously demonstrated in the HIS4 promoter(30) .
The additive and independent activation of LEU4 through
UAS, GCE-A, and GCE-B is unusual since in many other
eukaryotic promoters upstream elements activate transcription
synergistically(31, 32, 33) . Two major
models, the cooperative DNA binding model and the simultaneous contact
model, have been proposed to explain synergistic
activation(34, 35) . In the simultaneous contact
model, multiple contacts with the transcription apparatus would have a
multiplicative effect. We would like to propose a variant of the
simultaneous contact model to explain the additive effect observed with
the LEU4 promoter. We envision that Leu3p and Gcn4p might each
facilitate the assembly of a different subcomplex prior to formation of
the final preinitiation complex. This idea is in agreement with recent
findings suggesting that the preinitiation complex might not be
assembled by the stepwise addition of individual components but that
some of the components might exist in
subcomplexes(36, 37) . The in vivo partners
of Leu3p and Gcn4p are not yet known. However, in vitro experiments have shown that Gcn4p can interact directly with RNA
polymerase II (38) and that Leu3p can interact with
TBP(39) . These results are consistent with the notion that the
two regulatory proteins interact with the transcription apparatus at
different stages of assembly. Also of interest in this context is the
relative arrangement of the Leu3 and Gcn4 proteins along the promoter
DNA. If we assume that the UAS
of LEU4, like
that of LEU2(11) , consists of two contact triplets 9
bp apart (center-to-center), then the downstream contact triplet
(centered on position -441) would be separated by approximately
two helical turns from the symmetrical center of GCE-A (assuming B form
DNA); GCE-A would be separated by about six helical turns from GCE-B.
This arrangement would allow the Leu3 and Gal4 proteins to bind to the
same face of the DNA double helix and might thus make simultaneous
contacts between the activators and components of the preinitiation
complex thermodynamically favorable.
As is the case for many yeast genes under general control, LEU4 has multiple sequences in its promoter that are homologous to the 5`-TGACTC-3` consensus sequence. However, only two out of at least four putative general control response elements are functional in vivo. This phenomenon seems to be a recurring theme in the general control system of yeast. Only two out of seven TGACTC-like sequences in the noncoding region of HIS3 are apparently functional(40) . Similarly, while most of the five general control repeats in the noncoding region of HIS4 are required for full derepression under starvation conditions, one repeat stands out in its significance(41, 42) . The one general control repeat present in the noncoding region of ARO7 does not support Gcn4p-mediated regulation even though it specifically binds Gcn4p in vitro(43) . It is not known why some Gcn4p-binding elements function in vivo while others do not; it has been suggested that additional features, e.g. chromatin structure, may be responsible for the differences in efficiency(43) . The two functional general control elements of LEU4 are not entirely equivalent. Mutating the GCE-B element not only reduces the starvation/surfeit ratio but also yields lower absolute values of LEU4 expression, suggesting that the GCE-B element is involved in basal level regulation of LEU4 also. Again, there are several prior examples of such dual functionality of Gcn4p-binding sites(40, 42, 44) .
The regulation of LEU4 expression by Leu3p--IPM, general amino acid
control, and the phosphate level is complemented and augmented by the
regulation of the activity of
-IPM synthase. Besides being subject
to feedback inhibition by leucine,
-IPM synthase is reversibly
inactivated by coenzyme A, an effect that appears to be correlated with
the control of acetyl-CoA utilization and that can be reversed or
prevented by a high energy charge(45) . We believe that at
least part of the physiological reason for this tight and diversified
control of the first step in leucine biosynthesis is the fact that
-IPM also acts as a co-activator of genes that are controlled by
Leu3p. The recent discovery that Leu3p-
-IPM is an important
regulator of the main ammonia-assimilating enzyme in yeast (the GDH1-encoded NADP
-dependent glutamate
dehydrogenase) strongly suggests that the yeast cell utilizes
-IPM
as a signal molecule to feed information from the periphery back to the
center of nitrogen anabolism(13) . Just how far the
-IPM-controlled network extends remains to be established.