©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Additive Activation of Yeast LEU4 Transcription by Multiple cis Elements (*)

(Received for publication, August 1, 1994; and in revised form, December 20, 1994)

Yuanming Hu Gunter B. Kohlhaw (§)

From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The LEU4 gene of Saccharomyces cerevisiae and the enzyme encoded by LEU4, alpha-isopropylmalate synthase, occupy a special position in amino acid metabolism. alpha-Isopropylmalate synthase catalyzes the first committed step in leucine biosynthesis. However, the reaction product alpha-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 alpha-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.


INTRODUCTION

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 (^1)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 (alpha-isopropylmalate synthase EC 4.1.3.12, [alpha-IPMS]) provide an interesting example of multiple controls. alpha-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 alpha-IPM, a product of the alpha-IPMS catalyzed reaction. When leucine is in short supply, diminished feedback inhibition of alpha-IPMS causes the alpha-IPM level to rise. alpha-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 alpha-IPM is present or absent. When alpha-IPM is absent, Leu3p is inert or acts as a repressor of transcription; incoming alpha-IPM then changes Leu3p from an inactive (repressive) to an active configuration(16, 17, 18) .

The pleiotropic mode of action of the Leu3pbulletalpha-IPM complex and the function of alpha-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 alpha-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.


MATERIALS AND METHODS

Strains and Media

The S. cerevisiae strains used here were XK25-1B (MATa ura3-52), XK53-31 (MATa ura3-52 leu1), XK147-2C (MATalpha ura3-52 leu1 LEU4), XK154-2D (MATa ura3-52 leu1 LEU4gcn4-101), XK154-8A (MATalpha ura3-52 leu1 LEU4 leu3-Delta2::HIS3), and XK154-5B (MATalpha ura3-52 leu1 LEU4gcn4-101 leu3-Delta2::HIS3). The Escherichia coli strains were TG1 (K12 Delta(lac-pro) supE hsdDeltaS/F` traDelta36 proABlacI^qlacZM15) and CJ236 (dut1 ung1 thi-1 relA1/pCJ105(CM^r)). YPD (1% yeast extract, 2% Bacto-peptone, and 2% glucose) and SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose) with various additions were used as indicated. YPD medium that has depleted inorganic phosphate was prepared as described by Rubin(24) . The E. coli strain TG1 was routinely used for DNA manipulations. Strain CJ236 was used for isolation of uracil-containing single-stranded (ss) DNA. E. coli cells were grown in L broth or 2 times YT media (25) with the addition of 100 µg/ml ampicillin where needed.

Site-directed Mutagenesis

The oligonucleotides used in this study are shown in Table 1. M13mp11/401-6 is a M13mp11 derivative containing a 786-bp XbaI-BamHI LEU4 promoter fragment. The BamHI site was newly created two codons downstream from the translational start of LEU4. For ss DNA production, CJ236 cells were infected with M13mp11/401-6 phage stock and shaken vigorously at 37 °C in the presence of 0.25 µg/ml uridine and 30 µg/ml chloramphenicol. Uracil-containing ss DNA was isolated and site-directed mutagenesis of the LEU4 promoter was performed according to the procedure supplied by Bio-Rad. Mutations were confirmed by sequencing following a United States Biochemical Corp. (Cleveland, OH) protocol.



LEU4`-`lacZ Fusion Plasmid Construction

Plasmid pSEYC102 was a gift from S. Emr, University of California, San Diego. For deletion constructs the M13mp11/401-6 phage DNA containing the LEU4 promoter (see above) was cut with restriction enzymes PvuII, StuI, RsaI, TaqI, or BsaAI, blunt ended with the Klenow fragment of DNA polymerase when necessary, then cut with BamHI. The isolated fragments were ligated to SmaI-BamHI-digested pSEYC102, giving rise to pYH1 (contains a 690-bp PvuII-BamHI promoter fragment), pYH2 (393-bp StuI-BamHI fragment), pYH3 (342-bp RsaI-BamHI fragment), pYH4 (326-bp PvuII-BamHI fragment), pYH5 (220-bp TaqI-BamHI fragment), and pYH6 (119-bp BsaAI-BamHI fragment). An additional series of fusion plasmids was constructed by performing site-directed mutagenesis on M13mp11/401-6, followed by ligation of the isolated mutant PvuII-BamHI fragments to SmaI-BamHI-digested pSEYC102 (plasmids pYH7-pYH15 and pYH17-pYH20). Plasmid pYH16 was constructed by excising a StuI-BamHI fragment containing a mutated GCE-B box from M13mp11/401-6 and ligating it to SmaI-BamHI-digested pSEYC102. In all of the fusion plasmids, the first two amino acid codons of LEU4 (Met-Val) were attached to the 10th codon of lacZ (Val) via a bridge of three codons (Arg-Asp-Pro) introduced by the newly constructed BamHI site.

Yeast Transformation, Cell Growth, and beta-Galactosidase Assays

The LEU4-lacZ fusion plasmids were used to transform strains XK147-2C, XK154-2D, XK154-8A, XK154-5B, XK53-31, or XK25-1B using a modified lithium acetate method(26) . Transformants were purified once, and single colonies were precultured in branched-chain amino acid surfeit medium (SD plus 2 mM leucine, 1 mM isoleucine, and 1 mM valine). Precultures were divided and used to inoculate both a surfeit and an amino acid starvation medium, the latter consisting of SD plus 0.2 mM leucine. The calculated starting OD was 0.05. Cells were incubated at 30 °C and harvested at an OD of 0.9 (surfeit medium mid-log phase) or 0.4-0.5 (starvation medium late log phase). beta-Galactosidase activity was measured as described previously with minor modifications(10) . Briefly, harvested cells were washed once with double-distilled H(2)O, once with Z buffer (100 mM sodium phosphate buffer, pH 7.0, 10 mM KCl, 1 mM MgSO(4), 25 mM beta-mercaptoethanol) and resuspended in Z buffer. Twenty µl of 0.1% SDS and 50 µl of chloroform were added to 1-ml aliquots of cell suspensions, and the mixture was vortexed for 30 s. After a 15-min incubation at 30 °C, 0.2 ml of an o-nitrophenyl-beta-D-galactopyranoside solution (4 mg/ml in Z buffer) were added, and the incubation was continued until visible color developed. The reaction was stopped by adding 0.5 ml of 1 M Na(2)CO(3). Cell debris was removed and the OD measured immediately. Specific activities were determined according to Miller(27) . To assay cells grown in low phosphate medium, plasmid-bearing strains were grown overnight in 10 ml of SD medium. Cells were washed with sterile distilled H(2)O, transferred to low phosphate YPD, and grown at 30 °C until an OD of about 1.8. To minimize the difference in residual phosphate concentration in the low phosphate YPD medium, the same batch of medium was used for all experiments. The harvested cells were assayed for beta-galactosidase activity as described above.

DNase I Footprinting

A partially purified preparation of yeast TATA-binding protein (yTBP) was a gift from Karen Arndt, Harvard University. DNase I was obtained from Boehringer Mannheim. The probe used for footprinting was a LEU4 promoter fragment, prepared by polymerase chain reaction, extending from position -343 to position -43. It was end-labeled with [P]ATP and purified using a Microcon spin column (Amicon, Inc.). The probe was cut with restriction enzymes HaeIII and DdeI, respectively, to obtain two individual fragments labeled at one end only. Fifteen ng of DNA were incubated with or without TBP in 25 mM Tris-HCl buffer, pH 8.0, containing 6.25 mM MgCl(2), 0.5 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol, 200 ng of poly(dGbulletdC), and 0.01% Nonidet P-40 in a total volume of 100 µl for 30 min at 23 °C. Then 0.25 units of DNase I were added. After 1 min at 23 °C, the reaction was stopped by adding 600 µl of precooled stopping solution (0.05 M potassium acetate plus 5 µg of tRNA dissolved in 588 µl of ethanol). The DNA that precipitated during 2 h at -70 °C was pelleted by centrifugation at 4 °C for 20 min. The pellet was washed once with ice-cold 90% ethanol and vacuum-dried. Three µl of loading solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol (FF)) were added, and the sample was heated at 90 °C for 5 min and chilled on ice. Samples were loaded on a 6% sequencing gel to resolve the DNA fragments. The gel was fixed, dried, and autoradiographed.


RESULTS

Mutational Analysis of the Leu4 Promoter Identifies Three Upstream Regulatory Elements

The promoter analysis was performed using plasmids in which normal and mutated LEU4 promoter sequences were fused to the E. coli lacZ gene (Fig. 1). The plasmid was of the ARS1-CEN4 type, assuring a consistently low copy number of approximately one copy/cell. The LEU4-lacZ fusion was constructed such that it retained no more than two 5` codons of the LEU4 open reading frame. This strategy eliminated the mitochondrial import signal of the native Leu4 protein(8) , thus avoiding complications that might arise from sequestering the Leu4-beta-galactosidase fusion protein in mitochondrial compartments.


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 beta-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 beta-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-Delta2 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 beta-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.

The UAS and GC Elements Activate LEU4 Expression Additively

To pursue the question of whether the LEU3- and GCN4-dependent controls operate additively or in some other fashion, we constructed an additional plasmid (pYH17) that was deficient in GCE-A and GCE-B. It was used, together with plasmids pYH1 (wild type LEU4 promoter), pYH7 (promoter lacking the UAS element), and pYH12 (promoter deficient in UAS, GCE-A, and GCE-B), to transform a strain (XK53-31) that was wild type with respect to LEU3 and GCN4 and also produced a normal, feedback-sensitive alpha-IPM synthase, thus allowing a normal response to leucine starvation and surfeit conditions. A comparison of the specific activities of beta-galactosidase in the transformed cells grown under conditions of leucine starvation and surfeit is shown in Fig. 3. It is evident that general control and Leu3p-mediated control of LEU4-lacZ are additive. Under the conditions of the experiment, there was a 3.1-fold activation from the UAS alone (plasmid pYH17). In the absence of UAS, but the presence of the two functional GCE elements, a 5.3-fold activation was observed (plasmid pYH7). When all elements were present, there was a 6.7-fold activation (plasmid pYH1). The two GCE elements also appear to act additively (Fig. 2B). The presence of both elements (plasmids pYH7, pYH10, pYH11, pYH15) resulted in a 4.4-fold activation, on the average. When the GCE-A element was mutated, there was an average activation of 2.6-fold (plasmids pYH8, pYH13, pYH14); when the GCE-B element was mutated, a 2.9-fold activation was observed (plasmid pYH9).


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 beta-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 beta-galactosidase activity under starvation conditions. UAS(L), intact UAS element; UAS(L), 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.



The Proximal LEU4 Promoter Region Contains One Functional TATA Element and a Bas2 Response Element

The two segments of the LEU4 promoter that, when deleted, led to basal level II or near zero expression (Fig. 2A, plasmids pYH5 and pYH6) were located in the downstream region of the promoter where one might expect to find TATA elements. This portion of the promoter was therefore subjected to DNase I footprinting and mutational analysis. The DNase I protection assay was performed with a purified preparation of yeast TATA box binding protein (yTBP). The footprint (Fig. 4) showed protection between positions -250 and -271 on the coding strand and between positions -250 and -270 on the noncoding strand. This region contains a sequence, 5`-TATATA-3`, that perfectly matches a TATA consensus sequence. The apparent K(d) for binding of yTBP, defined as the concentration of yTBP required for half-maximal interaction, was approximately 5 nM. This value is well within the range of specific binding established earlier for yeast TFIID(29) . In addition to the TATA box region, an element that is 67% identical with the Bas2 response element (BRE) of the HIS4 promoter (20) was identified between positions -135 and -152 of the LEU4 promoter (5`-GAAAAATAACCAATAAAT-3`; noncoding strand). To explore the functional significance of both the TATA box and the potential BRE, we generated extensive mutations in both regions. An appropriate strain (XK25-1B) was then transformed with plasmids carrying either wild type or mutant element sequences in the promoter of a LEU4-lacZ fusion. The specific beta-galactosidase activity in the transformed cells (Table 2) demonstrated that the TATA element is essential for the expression of the LEU4-lacZ gene since destruction of the TATA box caused an almost total loss of promoter activity. Mutation of the BRE caused a reduction of promoter efficiency by about 40%. Simultaneous destruction of TATA and BRE had the same effect as mutation of TATA alone. To examine the significance of the -152 to -132 region for basal control of LEU4, we measured the LEU4-lacZ expression from wild type and mutated promoters as a function of the phosphate concentration in the medium. The results indicated the following (data not shown): (i) LEU4-lacZ expression was affected by the phosphate concentration. Under the conditions of the experiment, increasing the inorganic phosphate concentration from a low, depleted level to 5 mM caused a decrease of about 15% when the wild type promoter was present (plasmid pYH1), and a decrease of about 45% when a truncated promoter lacking UAS, GCE-A and -B, and the TATA box (plasmid pYH5) was used. (ii) The phosphate response was lost when LEU4-lacZ expression was directed by a promoter whose BRE was eliminated either by truncation (plasmid pYH6) or by site-specific mutagenesis (plasmid pYH19). (iii) Under phosphate sufficiency, the LEU4-lacZ expression level obtained with wild type promoter (pYH1) dropped to about half when the BRE region was mutated (plasmid pYH19). We conclude that LEU4 expression is subject to both phosphate and basal level controls. The fact that both controls were absent whenever a Bas2 response element was missing strongly suggests that both are brought about by the Bas2 protein.


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.






DISCUSSION

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-alpha-IPM, general amino acid control, and the phosphate level is complemented and augmented by the regulation of the activity of alpha-IPM synthase. Besides being subject to feedback inhibition by leucine, alpha-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 alpha-IPM also acts as a co-activator of genes that are controlled by Leu3p. The recent discovery that Leu3p-alpha-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 alpha-IPM as a signal molecule to feed information from the periphery back to the center of nitrogen anabolism(13) . Just how far the alpha-IPM-controlled network extends remains to be established.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant GM15102. This is Journal Paper No. 14364 of the Purdue University Agricultural Research Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 317-494-1616; Fax: 317-494-7897.

(^1)
The abbreviations used are: UAS, upstream activating sequence; URS, upstream repressing sequence; alpha-IPMS, alpha-isopropylmalate synthase; GCE, Gcn4p-binding elements; ss, single-stranded; bp, base pair(s); yTBP, yeast TATA-binding protein; BRE, Bas2 response element.


ACKNOWLEDGEMENTS

We thank Karen Arndt, Harvard University, for a gift of yeast TBP and Scott Emr, University of California, San Diego, for plasmid pSEYC102.


REFERENCES

  1. Guarente, L. (1987) Annu. Rev. Genet.21, 425-452 [CrossRef][Medline] [Order article via Infotrieve]
  2. Struhl, K. (1989) Annu. Rev. Biochem. 58, 1051-1077 [CrossRef][Medline] [Order article via Infotrieve]
  3. Luche, R. M., Sumrada, R., and Cooper, T. G. (1990) Mol. Cell. Biol. 10, 3884-3895 [Medline] [Order article via Infotrieve]
  4. Lopes, J. M., Schulze, K. L., and Yates, J. W. (1993) J. Bacteriol. 175, 4235-4238 [Abstract]
  5. Baichwal, V. R., Cunningham, T. S., Gatzek, P. R., and Kohlhaw, G. B. (1983) Curr. Genet. 7, 369-377
  6. Chang, L. L., Cunningham, T. S., Gatzek, P. R., Chen, W. J., and Kohlhaw, G. B. (1984) Genetics 108, 91-106 [Abstract/Free Full Text]
  7. Beltzer, J. P., Chang, L. L., Hinkkanen, A. E., and Kohlhaw, G. B. (1986) J. Biol. Chem. 261, 5160-5167 [Abstract/Free Full Text]
  8. Beltzer, J. P., Morris, S. R., and Kohlhaw, G. B. (1988) J. Biol. Chem. 263, 368-374 [Abstract/Free Full Text]
  9. Peters, M. H., Beltzer, J. B., and Kohlhaw, G. B. (1990) Arch. Biochem. Biophys. 276, 294-298 [Medline] [Order article via Infotrieve]
  10. Bai, Y., and Kohlhaw, G. B. (1991) Nucleic Acids Res. 19, 5991-5997 [Abstract]
  11. Remboutsika, E., and Kohlhaw, G. B. (1994) Mol. Cell. Biol. 14, 5547-5557 [Abstract]
  12. Friden, P., and Schimmel, P. (1988) Mol. Cell. Biol. 8, 2690-2697 [Medline] [Order article via Infotrieve]
  13. Hu, Y., Cooper, T. G., and Kohlhaw, G. B. (1995) Mol. Cell. Biol. 15, 52-57 [Abstract]
  14. Falco, S. C., Dumas, K. S., and Livak, K. J. (1985) Nucleic Acids Res. 13, 4011-4027 [Abstract]
  15. Petersen, J. G. K., and Holmberg, S. (1986) Nucleic Acids Res. 14, 9631-9651 [Abstract]
  16. Brisco, P. R. G., and Kohlhaw, G. B. (1990) J. Biol. Chem. 265, 11667-11675 [Abstract/Free Full Text]
  17. Zhou, K., and Kohlhaw, G. B. (1990) J. Biol. Chem. 265, 17409-17412 [Abstract/Free Full Text]
  18. Sze, J-Y., Woontner, M., Jaehning, J. A., and Kohlhaw, G. B. (1992) Science 258, 1143-1145 [Medline] [Order article via Infotrieve]
  19. Hinnebusch, A. G. (1988) Microbiol. Rev. 52, 248-273
  20. Arndt, K., and Fink, G. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8516-8520 [Abstract]
  21. Tice-Baldwin, K., Fink, G. R., and Arndt, K. T. (1989) Science 246, 931-935 [Medline] [Order article via Infotrieve]
  22. Daignan-Fornier, B., and Fink, G. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6746-6750 [Abstract]
  23. Fascher, K. D., Schmitz, J., and Hörz, W. (1990) EMBO J. 9, 2523-2528 [Abstract]
  24. Rubin, G. M. (1974) Eur. J. Biochem. 41, 197-202 [Medline] [Order article via Infotrieve]
  25. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) in Basic Methods in Molecular Biology , pp. 250 and 366, Elsevier Science Publ. Co., New York
  26. Gietz, D., Jean, A. S., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425 [Medline] [Order article via Infotrieve]
  27. Miller, J. H. (1972) in Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  28. Arndt, K. T., Styles, C., and Fink, G. R. (1987) Science 237, 874-880 [Medline] [Order article via Infotrieve]
  29. Hahn, S., Buratowski, S., Sharp, P. A., and Guarente, L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5718-5722 [Abstract]
  30. Pellman, D., Mclaughlin, M. E., and Fink, G. R. (1990) Nature 348, 82-85 [Medline] [Order article via Infotrieve]
  31. Mösch, H.-U., Graf, R., and Schmidheini, T. (1990) EMBO J. 9, 2951-2957 [Abstract]
  32. Ponglikitmongkol, M., Whit, J. H., and Chambon, P. (1990) EMBO J. 9, 2221-2231 [Abstract]
  33. Oshima, H., and Simons, S. S., Jr. (1993) J. Biol Chem. 268, 26858-26865 [Abstract/Free Full Text]
  34. Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1989) Cell 57, 443-448 [Medline] [Order article via Infotrieve]
  35. Emami, K. H., and Carey, M. (1992) EMBO J. 11, 5005-5012 [Abstract]
  36. Koleske, A. J., and Young, R. A. (1994) Nature 368, 466-469 [CrossRef][Medline] [Order article via Infotrieve]
  37. Kim, Y-J., Björklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608 [Medline] [Order article via Infotrieve]
  38. Brandl, C. J., and Struhl, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2652-2656 [Abstract]
  39. Remboutsika, E. (1994) Transcriptional Regulator Leu3p of Yeast: Modular Architecture and Function , Ph. D. Thesis, Purdue University, West Lafayette, IN
  40. Struhl, K., and Hill, D. E. (1987) Mol. Cell. Biol. 7, 104-110 [Medline] [Order article via Infotrieve]
  41. Donahue, T. F., Daves, R. S., Lucchini, G., and Fink, G. R. (1983) Cell 32, 89-98 [Medline] [Order article via Infotrieve]
  42. Lucchini, G., Hinnebusch, A. G., Chen, C., and Fink, G. R. (1984) Mol. Cell. Biol. 4, 1326-1333 [Medline] [Order article via Infotrieve]
  43. Schmidheini, T., Mösch, H. U., Graf, R., and Braus, G. H. (1990) Mol. & Gen. Genet. 224, 57-64
  44. Mösch, H., Scheier, B., Lahti, R., Mäntsälä, P., and Bruss, G. H. (1991) J. Biol. Chem. 266, 20453-20456 [Abstract/Free Full Text]
  45. Hampsey, D. M., and Kohlhaw, G. B. (1981) J. Biol. Chem. 256, 3791-3796 [Abstract/Free Full Text]

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