JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Nov. 1999, p. 7052–7064 Vol. 181, No. 22

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Synergistic Operation of the CAR2 (Ornithine Transaminase)Promoter Elements in Saccharomyces cerevisiae

HEUI-DONG PARK,1 STEPHANIE SCOTT,2 RAJENDRA RAI,2 ROSEMARY DORRINGTON,2†AND TERRANCE G. COOPER2*

Department of Food Science and Technology, Kyungpook National University, Taegu 702-701, Korea,1 andDepartment of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 381632

Received 7 July 1999/Accepted 7 September 1999

Dal82p binds to the UISALL sites of allophanate-induced genes of the allantoin-degradative pathway andfunctions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are respon-sible for nitrogen catabolite repression-sensitive gene expression. CAR2, which encodes the arginine-degrada-tive enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can bemodestly induced by the allantoin pathway inducer. The dominant activators of CAR2 transcription have beenthought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observationsprompted us to investigate the structure of the CAR2 promoter with the objectives of determining whether othertranscription factors were required for CAR2 expression and, if so, of ascertaining their relative contributionsto CAR2’s expression and control. We show that Rap1p binds upstream of CAR2 and plays a central role in itsinduced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogueoxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is inducedwhen cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediatelydownstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike allother known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3pand Gat1p, which occurs only in the presence of an inducer. The observations reported suggest that CAR2 geneexpression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p beingbalanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is thentipped in the direction of expression by the presence of the inducer. The formal structure of the CAR2 promoterand its operation closely follow the model proposed for CAR1.

All nitrogen catabolite repression (NCR)-sensitive genes inSaccharomyces cerevisiae have GATA sequences in their pro-moters, and their expression is dependent upon one or both ofthe GATA family transcriptional activators Gln3p and/orGat1p (also called Nil1p) (for a brief review of the GATAfactor field, see the introductions of references 7 and 52).Gln3p has been shown to bind to the GATA sequences, andboth Gln3p and Gat1p have the ability to activate transcriptionof a reporter gene in a lexAp-tethering assay system (7, 52).Expression of the genes encoding components of the arginine-and allantoin-degradative pathways depends upon Gln3p andGat1p working synergistically with pathway-specific transcrip-tion factors (47, 61). Arginine and allantoin pathway geneexpression is also subject to induction in response to the pres-ence of small signal metabolites (8, 9, 23, 37–40, 59, 60). In thecase of the arginine pathway genes (CAR genes), the inducer isarginine (59) and the transcription factors involved are Ar-gRIp, ArgRIIp, ArgRIIIp, and Mcm1p. For the allantoin path-way genes (DAL and DUR genes), the inducer is allophanateor its nonmetabolized analogue, oxalurate (OXLU), and theassociated transcription factors are Dal82p and Dal81p (8, 9).

The CAR1 promoter has been extensively studied both bio-chemically and genetically (2–4, 11, 12, 14, 18, 23, 25, 59). Itconsists of up to 14 cis-acting sites, which bind nine trans-acting

factors, Rap1p, Abf1p, Gln3p, Gat1p, ArgRIp, ArgRIIp,ArgRIIIp, Mcm1p, and Ume6p (3, 10, 13, 19, 27–29, 31, 34–36,42–44, 47, 51, 58). Proteins that bind to the Rap1 and Abf1sites and a GC-rich sequence are necessary to achieve highlyinduced transcription levels of CAR1 (27–29, 47). The action ofthese strong, constitutively acting transcription factors is an-tagonized by the equally strong negative action of Ume6p (alsocalled Car80p) bound to the URS1 site (1, 3, 42, 47–51). Themetabolite-responsive promoter elements are much weakerthan those just cited (47) and have been suggested to tip thebalance either toward expression when the inducer is presentand a repressing nitrogen source is absent or toward quies-cence when arginine is absent and/or a repressive nitrogensource such as glutamine is present (47, 50, 51). Three cis-acting sites, which are associated with the ArgRI, ArgRII,ArgRIII, and Mcm1 proteins, mediate arginine-dependent in-duction (10, 11, 13, 14, 18, 19, 34–36). NCR-sensitive CAR1expression, on the other hand, is mediated by two GATAsequences associated with Gln3p and Gat1p (47).

Although arginase and ornithine transaminase (encoded byCAR1 and CAR2) catalyze contiguous enzyme reactions in thearginine-degradative pathway of S. cerevisiae, production of thetwo enzymes is regulated somewhat differently (37–40, 59, 60).While arginine induces production of both arginase and orni-thine transaminase, only arginase production is NCR sensitive(14, 37–40, 59, 60). Production of ornithine transaminase, onthe other hand, is induced by allophanate, the last intermediateof the allantoin-degradative pathway, while CAR1 expression isnot (23). Unlike that of CAR1, the CAR2 promoter has notbeen characterized.

The allantoin pathway gene promoters appear to be simpler

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Tennessee, Memphis, TN 38163.Phone: (901) 448-6175. Fax: (901) 448-8462. E-mail: tcooper@utmem.edu.

† Present address: Department of Biochemistry and Microbiology,Rhodes University, Grahmstown 6140, South Africa.

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than those in the arginine pathway (8, 9). For example, theDAL7 promoter contains three types of sites, each of which isassociated with a different type of regulation. Multiple GATAsequences mediate NCR-sensitive gene expression that de-pends upon Gln3p and Gat1p (7–9, 51). DAL7 expression isdown regulated by the action of Dal80p, which antagonizestranscriptional activation, mediated by Gln3p and Gat1p (9).Finally, inducer-dependent (allophanate or its nonmetabolizedanalogue OXLU) expression depends upon the UISALL site;Dal82p binds to this site and Dal81p functions in associationwith it (8, 9, 17). A notable similarity exists in the formalorganizations of the CAR and DAL promoters; the action oftranscriptional activators is antagonized by repressor proteins,and the balance is shifted by the inducer-responsive transcrip-tion factor (11a, 47).

In all cases studied so far, UISALL and Dal82p function onlyin association with the GATA factors Gln3p and Gat1p (9).Further, genetic data have raised the possibility that Gln3pand/or Gat1p may interact directly with Dal82p (57). A UISALLsite placed adjacent to a mutated GATA-containing UASNTRsite will suppress its mutant phenotype. This suppression de-pends upon the presence of wild-type Gln3p and Dal82p andcorrelates with Dal82p binding to UISALL (57). It does not,however, depend upon Dal81p or an inducer. Dal81p does notappear to bind to DAL promoters (4a, 56).

This work has three objectives, namely, to (i) identify thepromoter elements of CAR2 responsible for allophanate-in-duced expression; (ii) determine whether the overall structureand operation of CAR2 is analogous to those features of CAR1,i.e., whether CAR2 contains strong, opposing positive and neg-ative control elements, with the metabolite-responsive ele-ments shifting the balance; and (iii) determine whether Dal82pcan function in association with global transcription factorsother than Gln3p and/or Gat1p, and if so, which ones. Thiswork does not address elements and proteins associated witharginine-dependent transcriptional activation. This aspect ofCAR2 gene regulation has been reported by Messenguy andDubois and their colleagues.

MATERIALS AND METHODS

Strains and media. The S. cerevisiae strain used for b-galactosidase assays wasTCY1 (MATa lys2 ura3), and the wild-type strain used for electrophoretic mo-bility shift assays (EMSAs) was W303-1A (MATa ade2-1 can1-100 his3-11,15leu2-3,112 trp1-1 ura3-1). Strains HEY6 and RDY4 are isogenic derivatives ofstrain TCY1 containing deletions of the DAL81 and DAL82 genes, respectively.Strain YLS91 (29) (kindly provided by David Shore) is a derivative of W303-1Acontaining two RAP1 genes; one gene is disrupted by LEU2 (which results in adeletion between the two BamHI sites at nucleotides 818 and ca. 2200), and theother has nucleotides 894 to 1584 deleted, which results in a deletion of aminoacids 44 to 303. The RAP1 DNA binding domain is located between residues 361and 596 of the 827-amino-acid protein (24). Therefore, the truncated protein stillretains the DNA binding sites (29). The Escherichia coli strain used for cloningwas HB101 (supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1).Yeast cultures used for Northern blot analysis and b-galactosidase assays weregrown in medium containing 0.17% yeast nitrogen base without amino acids orammonium sulfate (YNB medium) and supplemented with 2% glucose and 0.1%arginine (induced condition) or 0.1% proline (noninduced condition). OXLUwas added to YNB-proline medium at a final concentration of 61 mg/ml for theCAR2 induction. The medium used for yeast extract preparations was YPD (1%yeast extract, 2% Bacto Peptone, 2% dextrose).

Northern blot analysis. Yeast cultures used for Northern blot analyses weregrown to the mid-log phase (40 to 60 Klett units). Total RNA was isolated by themethod of Carlson and Botstein (5). Poly(A)1 RNA was isolated with oligo(dT)-cellulose (Pharmacia Amersham) columns and resolved on 1.4% agarose–form-aldehyde gels (49). The resolved RNAs were transferred to GeneScreen Plusnylon 66 (Dupont, NEN Research Products) membranes. The membranes werethen hybridized to double-stranded DNA probes made radioactive by the ran-domly primed labeling method (Boehringer Mannheim). The procedures fol-lowed for hybridization and washing were those described in the GeneScreenPlus protocol manual.

PCR. PCR was carried out with BamHI-digested plasmid pRS427 containingthe CAR2 gene in the BamHI site of plasmid pBR322 as the template (48a). PCR

primers used in this study were designed to amplify the CAR2 59 regulatoryregion shown in each construct. The PCR mixture consisted of 0.5 mg of templateDNA, 100 pmol of each primer, 2.5 U of Taq DNA polymerase, 0.25 mMconcentrations of each deoxynucleoside triphosphate, 10 mM Tris-Cl (pH 8.3),50 mM KCl, and 2.5 mM MgCl2. The PCR cycle program for DNA amplificationconsisted of one cycle of 94°C (5 min) and 30 cycles of 94°C (1 min), 37°C (1min), and 72°C (3 min) followed by one cycle of 72°C (5 min). The PCR productswere purified with Sephadex G25 spun columns and subjected to enzyme diges-tion.

Plasmid constructions. Plasmid constructions for the CAR2 59 deletion anal-ysis were carried out by PCR. PCR products contained CAR2 DNA sequencesbetween various 59 upstream positions (2634, 2530, 2470, 2420, 2380, 2350,2302, 2251, 2233, 2205, 2177, 2165, and 2120) and position 13 as well asSalI and BamHI sites at the 59 and 39 termini of the products, respectively. Thefragments were digested with SalI and BamHI and were cloned into SalI andBamHI sites of plasmid pLG669Z, described in detail by Guarente et al. (20–22).

Plasmid pHP52, used to assay CAR2 upstream activation sequence (UAS)-mediated reporter gene expression, is identical to plasmid pNG15 (27), exceptthat it contains a 2mm yeast replication origin instead of ARS1, and was con-structed as follows. (i) The 2.2-kb EcoRI DNA fragment, containing a 2mmreplication origin, was cloned into plasmid pNG15 which had been partiallydigested with EcoRI to delete the TRP1 (also called ARS1)-containing fragment,to yield plasmid pHP51. (ii) The NcoI restriction site downstream of the lacZgene in plasmid pHP51 was destroyed by NcoI partial digestion and Klenowpolymerase reaction to produce plasmid pHP52. (iii) Synthetic oligonucleotides,containing putative CAR2 UAS elements, were cloned into the SalI and EagIsites of the heterologous expression vector pHP52. The core promoter containedin plasmid pNG15 is a modified fragment (14 to 21100) from the CYC1 gene.The cloning was done with a polylinker that replaced the CYC1 sequence from2228 to 2700.

Plasmid pHP200 was constructed to assay the effects of mutating one or moreof the cis-acting elements in the context of a full-length CAR2 promoter. PCRprimers were designed to amplify CAR2 DNA sequences between positions2126 and 13. SalI and BamHI restriction sites (11 bp apart) were added to the59 terminus, and a BglII site was added to the 39 terminus. The sequence59-CCCTTGCCCTTAGCGGCTGACTGGCT-39 was changed to 59-CCCTATAGTCGACCGGCTGGGATCCT-39. The PCR product was digested with SalIand BglII and cloned into plasmid pLG669Z that had been digested with SalI andBamHI; the BamHI-BglII ligation destroyed the BamHI site at the junction andfused the CAR2 sequence from 2126 to 13 in frame to lacZ. These stepsproduced the vector plasmid pHP200. PCR amplification was then used togenerate DNA fragments containing wild-type and mutant alleles of the CAR2 59regulatory region between positions 2302 and 2127. These fragments were thencloned into plasmid pHP200 digested with SalI and BamHI, reassembling afull-length CAR2 promoter fused to lacZ. The only substitutions of wild-typesequences were those necessary to introduce the SalI restriction site. This sitewas situated downstream of the TATA box to avoid any changes in the putativeUAS region of the plasmid. b-Galactosidase activities were measured with trans-formants which contained the set of plasmids constructed in this way. All theDNA fragments derived from synthetic oligonucleotides and PCR products wereverified in the recombinant plasmids by sequence analyses (53, 54). Other tech-niques, including DNA manipulation and DNA gel electrophoresis, were asdescribed in detail by Sambrook et al. (45).

EMSA. The sequences of synthetic oligonucleotides used as DNA probes andcompetitors are shown in Fig. 1. Cultures of S. cerevisiae W303-1A and YLS91,used for the preparation of crude extracts, were grown in YPD medium. Theprotocols for extract preparation and EMSAs were as described by Luche et al.(31) and Kovari et al. (29). Within experimental error, the lanes of the gels(depicted as individual autoradiographs) contained the same amounts of theDNA probes.

Yeast and bacterial transformation. Yeast strains were transformed with lith-ium acetate by the method of Ito et al. (26). E. coli HB101 was transformed bythe Tschumper and Carbon modification (55) of the Mandel and Higa method(33).

b-Galactosidase assay. Assays of b-galactosidase activities were performed induplicate and also from duplicate or triplicate independent yeast transformantsby using the procedures and precautions described in detail earlier (21, 27, 47).Data from repeated experiments generally varied less than 20%, and data fromduplicate assays varied less than 5%. The patterns of reporter gene expressionobserved within each experiment (the results of one experiment are representedin each figure) were invariant upon repetition of the experiment, unless other-wise noted. However, as noted earlier, quantitative comparisons of data areprudent only when all of the results being compared derive from a single exper-iment (i.e., data from within a figure). Enzyme activities are expressed in unitsdefined by Miller (41) but were based on 10 ml of culture rather than 1 ml.

RESULTS

Induced CAR2 expression. We first established the CAR2baseline induction pattern using Northern blot analysis.Steady-state levels of CAR1 mRNA (our positive control gene)

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were much higher in cells provided with arginine as the nitro-gen source than with proline, i.e., CAR1 expression was highlyinducible. (Fig. 2, lanes A and B). Compared to those of CAR1,CAR2 mRNA levels are less responsive to arginine largely dueto the fact that substantial basal-level CAR2 expression occurswhen proline is provided as the sole nitrogen source (lanes Cand E). CAR2 expression was induced by OXLU but to a muchsmaller degree than when arginine was used as the inducer(Fig. 2, lanes C and D).

5* deletion analysis of the CAR2 upstream region. We begandissection of the CAR2 promoter by constructing a CAR2-lacZfusion plasmid (pHP120) (see Materials and Methods) con-taining 634 bp of the upstream region. This plasmid supportedtwo- and eightfold-higher levels of b-galactosidase productionin response to the presence of OXLU and arginine, respec-tively (Fig. 3). A series of deletions removing nucleotides 2634to 2302 (plasmids pHP120 to pHP126) had little effect onb-galactosidase production, suggesting that this region was notdemonstrably required for regulated CAR2 expression (Fig. 3).Deletion of nucleotides 2302 to 2251 resulted in three-, four-,and twofold decreases in basal, proline- and OXLU-induced,and arginine-induced levels of lacZ expression, respectively(Fig. 3, compare plasmids pHP126 and pHP127). Removal ofthe next 18 nucleotides to position 2233 resulted in further10-, 11-, and 7-fold decreases in lacZ expression, respectively(Fig. 3, plasmid pHP128). In plasmid pHP128, only low levels

of arginine-induced reporter gene expression were observed.Deletion of 28 additional bases to position 2205 (plasmidpHP129) resulted in the loss of the remaining arginine-depen-dent b-galactosidase production. The same result was observedwith the three subsequent deletions, the last of which (plasmidpHP132) lacked all sequences 59 of position 2120; the TATAsequence of CAR2 is situated at position 2134. These datademonstrate that the region between CAR2 positions 2302and 2233 contains elements that are important for high-levelexpression when arginine is used as the nitrogen source as wellas for the more modest level of expression when OXLU ispresent.

Demonstration of a putative Abf1p binding to the regionfrom 2302 to 2233 of CAR2. Inspection of the CAR2 upstreamregion from 2302 to 2233 revealed sequences homologous toABF1, RAP1, UISALL and UASARG sites (Fig. 4). Notablyabsent were GATAA-containing sequences homologous toUASNTR. A priori, the presence of sequences homologous toknown transcription factor binding sites is not sufficient reasonto conclude that transcription factors bind to them and medi-ate CAR2 transcription. Therefore, EMSAs were performed toascertain whether CAR2 promoter fragments containing thesehomologous sequences were indeed able to bind protein.When radioactively labeled CAR2 fragment HD189 (positions2313 to 2271) was incubated with a wild-type yeast crude cellextract (see Materials and Methods), two retarded specieswere observed (Fig. 5, lane B); no retarded species was ob-served when extract was omitted from the EMSA reactionmixture (lane A). To assess the binding specificity, we used amutant allele of fragment HD189 in which sequences homol-ogous to an ABF1 binding site were altered in a way previouslyshown to destroy Abf1p binding (15, 16, 28). The slower-mi-grating DNA-protein complex was lost when the mutant pro-moter fragment was used as the probe (Fig. 5, lane C). Thesedata suggested that only this slower-migrating species was po-tentially associated with Abf1p binding. The identity of theprotein(s) in the more rapidly migrating complex in lanes Band C remains unknown.

To determine whether the more slowly migrating speciespossessed a migration rate similar to that of a legitimateAbf1p-DNA complex, we used a well-characterized, similarlysized DNA fragment containing the HMRE ARS1 B domain

FIG. 1. Oligonucleotides used as radioactive probes and competitors inEMSAs. Sequences homologous to Abf1p, Rap1p, and Dal82p binding sites areindicated with brackets. Mutations in each site, here and in all other figures, areshown as lowercase letters. Numbers in the figure indicate the positions of thebases whose coordinates are given. mt, mutant.

FIG. 2. Steady-state levels of CAR1 and CAR2 mRNAs in S. cerevisiae grownon various nitrogen sources. Poly(A)1 RNA was prepared from S. cerevisiaeS1278b cells grown in YNB media containing proline (PRO) or arginine (ARG).OXLU, a gratuitous inducer, was added to YNB-proline medium(PRO1OXLU) at a final concentration of 61 mg/ml for the CAR2 induction. Thesame designations for growth conditions are used throughout this work. HistoneH4 mRNA served as a control for Northern blot loading and transfer efficiencies.

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(15, 16, 28), which was used as an EMSA probe in the analysisof the CAR1 promoter (28) (Fig. 5, lanes D to F). The wild-type HMRE fragment yielded a retarded species with a mo-bility similar to the one observed with the wild-type CAR2

fragment (lane E); as expected, retardation was not observedwith the mutant HMRE fragment (lane F).

Competition EMSAs further substantiated the specificity ofthe DNA-protein complex. In the first of these experiments,CAR2 fragment HD189 was used as the radioactive probe andits ability to form a retarded complex was assayed in the pres-ence of increasing amounts of nonradioactive HMRE ARS1 Bdomain DNA as the competitor (Fig. 6, left panel). The lowestconcentration of competitor DNA successfully competed withthe CAR2 fragment probe (Fig. 6, left panel, lanes A to H). Incontrast, when a mutant allele of the HMRE fragment wasused in the assay, no competition was observed (Fig. 6, leftpanel, lanes H to O). This experiment was then repeated withthe probe and competitor reversed, i.e., with the HMRE frag-ment as the probe and the CAR2 fragment as the competitor.As shown in the right panel of Fig. 6, the CAR2 fragment wasonly minimally able to compete with the HMRE DNA frag-ment for protein binding (lanes A to H). This is not a surpris-ing result given the dramatic effectiveness of HMRE as acompetitor in the left panel of the figure. Together, the data inFig. 5 and 6 suggest that a CAR2 fragment containing a se-quence from positions 2313 to 2271 binds to Abf1p. Note thata more rapidly migrating species in panel A (for which theCAR2 DNA probe was used) was equally competed away byincreasing amounts of wild-type or mutant HMRE DNA, ar-guing that this complex does not depend upon sequences ho-mologous to Abf1p binding sites. It is, however, unlikely to benonspecific binding of protein to DNA because the band wasnot observed when HMRE DNA was used as the radioactiveprobe.

Demonstration of putative Rap1p binding to the regionfrom 2302 to 2233 of CAR2. We next investigated a CAR2sequence contained in DNA fragment HD190 (CAR2 positions

FIG. 3. 59 deletion analysis of the CAR2 upstream region. DNA fragments containing the CAR2 regulatory region as well as SalI and BamHI restriction sites at the59 and 39 termini, respectively, were amplified from CAR2 DNA by PCR. The PCR products were digested with SalI and BamHI and cloned into the SalI and BamHIsites of the lacZ expression vector pLG669Z (20–22). The set of plasmids was then used to transform strain TCY1 for analysis. Boxes at the top of the figure representsequences that are homologous to various transcription factor binding sites. T’s indicate the positions of TATA sequences. Numbers at the left of each insert indicatethe 59 terminus of the remaining CAR2 DNA in the CAR2-lacZ fusion plasmids. Coordinates are indicated relative to the position of the translation start site in thisand all subsequent figures.

FIG. 4. CAR2 sequences homologous to the binding sites for Abf1p, Rap1p,and Dal82p.

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2282 to 2247) which was homologous to known Rap1p DNAbinding sites (Fig. 4). One major and one minor retardedspecies were observed in the EMSA when extract from thewild-type strain W303-1A was used as the source of protein

(Fig. 7, lane B). When the assay was repeated with an extractderived from the rap1 mutant strain YLS91 (carrying a trun-cated, but binding-competent form of Rap1 [6, 24, 29, 30, 46]),the major retarded species migrated to a new location justbelow the minor species observed in lane B (Fig. 7, lane C).When a mutant CAR2 DNA probe (fragment HD192, in whichthe Rap1p site-homologous sequence is mutated) was used inthe assay, little if any of the more slowly migrating DNA-protein complex present in lane B was observed (Fig. 7, laneD). The more rapidly migrating species in lane B was alsoobserved in lane D, arguing that it was not associated with themutated sequence in fragment HD192. The small amount ofDNA-protein complex in lanes C and D that possessed thesame mobility as the major complex observed in lane B isunlikely to be Rap1p specific because it was also observed inlane C, which did not contain full-length Rap1p. We comparedthe data shown in Fig. 7, lanes A to D, with that obtained witha similarly sized, well-characterized TEF2 DNA probe previ-ously documented to contain a Rap1p binding site (6, 24, 29,30, 46). The pattern of data obtained in this control experiment(Fig. 7, lanes E to H) was the same as that observed when theCAR2 fragment was used (lanes A to D). The EMSA using theTEF2 probe also exhibited the nonspecific protein-DNA com-plexes seen in lanes B to D.

We next assessed the ability of the TEF2 and CAR2 frag-ments to serve as competitors of one another’s protein binding.At the time this experiment was performed, the Rap1p bindingsite contained in the TEF2 DNA fragment was the strongestone known (6, 24, 29, 30, 46). As shown in the left panel of Fig.8, the TEF2 DNA fragment was unable to serve as an effectivecompetitor of the CAR2 fragment for DNA binding (lanes A toH). In the reverse situation, i.e., when the TEF2 fragment wasused as the probe and the CAR2 fragment was used as thecompetitor, strong competition was observed (Fig. 8, right pan-el). In fact, even at the lowest concentration used, the wild-type

FIG. 5. Protein binding to ARS1 and CAR2 DNA fragments. DNA fragmentslabeled with 32P at their 59 ends and containing the HD189 and ARS1 sequencesindicated in Fig. 1 were used as probes. Yeast cell extract (strain W303-1A) wasomitted from the reaction mixture resolved in lanes A and D. All reactionmixtures contained a 200-fold excess of sheared calf thymus DNA by sonicationas a nonspecific competitor. 2EXT, minus extract, W.T., wild type; mut, mutant.

FIG. 6. Competition between CAR2 and ARS1 DNA fragments. An oligonucleotide covering CAR2 positions 2313 to 2271 (left) or ARS1 positions 748 to 780 andflanked by SalI and EagI restriction sites (right) was labeled at its 59 end with 32P and used as the probe. Competitor DNA was omitted in the reaction mixtures resolvedin lanes G and I. The reaction mixtures in lanes A through F contained decreasing amounts of the unlabeled ARS1 (left) or CAR2 (right) oligonucleotide as a competitor(Comp. and COMP). The reaction mixtures in lanes J through O contained increasing amounts of the unlabeled ars1 mutant (mut.) (left) or car2 mutant (right)oligonucleotides; the amounts that were used are indicated in micrograms (g). The remaining conditions and procedures used in the experiment were as described inthe legend to Fig. 5. Strain W303-1A was the source of the crude cell extract (EXT).

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CAR2 DNA fragment (HD190) effectively competed with theTEF2 probe (Fig. 8, right panel, lanes A to H). However, whenthe putative Rap1p binding site contained in the CAR2 frag-ment was mutated, the fragment was no longer able to serve asthe competitor. As with Abf1p, these data support the conten-tion that the CAR2 fragment from positions 2282 to 2247contains a putative Rap1p binding site.

Demonstration of putative Dal82p binding to the regionfrom 2302 to 2233 of CAR2. The last CAR2 sequence weinvestigated was one homologous to UISALL elements that aresituated upstream of allantoin pathway genes (Fig. 4). Theseelements are Dal82p binding sites that mediate induction ofDAL and DUR gene expression when allophanate or its ana-logue, OXLU, is present (9, 17). To determine whether Dal82pbound to the CAR2 UISALL-homologous sequence (2272 to2211), CAR2 fragment RD43 (Fig. 1) was incubated withextracts derived from an E. coli culture transformed with a T-7expression vector plasmid (pRD4) or one containing a T-7–DAL82 fusion (plasmid pRD41) (Fig. 9, lanes D to F). Nosignal was observed with extract derived from a transformantcontaining plasmid pRD4 alone (lane E). In contrast, severalDNA-protein complexes were observed with extract derivedfrom cells containing the plasmid bearing the T-7–DAL82 fu-sion (Fig. 9, lane F). Two of these complexes (indicated byarrows) were specific to the presence of Dal82p. The Dal82p-specific signal with the fastest mobility has been previouslyshown to derive from a Dal82p degradation product (17). Thesource of the weak nonspecific signal observed in the lowest-mobility complex is unknown (Fig. 9, lanes E and F). DAL7DNA fragment RD72X, which contains a well-characterizedDAL UISALL element (17), was used in a control experimentto identify the relative mobility of a bona fide UISALL-Dal82pcomplex (Fig. 9, lanes A to C). In this experiment, the major

FIG. 7. Wild-type and truncated Rap1p binding to CAR2 and TEF2 DNAfragments. DNA fragments 32P labeled at their 59 ends and containing theHD190 and HD192 sequences indicated in Fig. 1 were used as probes. Yeast cellextract (EXT) was omitted from the reaction mixtures resolved in lanes A and E.A wild-type (strain W303-1A) extract (W.T.) was used in lanes B, D, F, and H.An extract from a rap1 truncation mutant strain (mut.) (YLS91) was used inlanes C and G (rap1D).

FIG. 8. Competition between CAR2 and TEF2 DNA fragments for protein binding. An oligonucleotide 32P labeled at its 59 end and covering CAR2 positions 2282to 2247 (left) or TEF2 positions 2449 to 2414 (right) was used as the probe. The reaction mixtures in lanes A through F contained decreasing amounts of the unlabeledTEF2 (left) or CAR2 (right) oligonucleotide as a competitor (Comp. and COMP). The reaction mixtures in lanes J through O contained increasing amounts of theunlabeled TEF2 mutant (mut.) (left) or CAR2 mutant (right) oligonucleotide. The remaining conditions and procedures used in the experiment were as described inthe legend to Fig. 6. EXT, extract; g, microgram amounts.

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complexes with the lowest and greatest mobilities were theonly ones that were Dal82p specific and migrated to positionssimilar to those observed in lanes D to F.

To further demonstrate that the major complex observed inFig. 9 derived from a bona fide Dal82p-CAR2 UISALL inter-action, we performed a competition EMSA using CAR2 wild-type (RD43) and mutant (RD44) fragments as competitors ofDAL7 DNA fragment JRD72X (Fig. 10). The wild-type frag-ment was an effective competitor of JRD72X DNA binding(lanes A to F). Fragment RD44, however, which contained amutated form of the CAR2 UISALL, was unable to effectivelycompete with DAL7 for binding to DNA (Fig. 10, lanes H toM). These data led to the conclusion that the CAR2 fragmentRD43 contains a sequence capable of binding Dal82p.

Participation of the putative Abf1p, Rap1p, and Dal82pbinding sites in transcription. The 70-bp CAR2 fragment(2303 to 2235), required for high-level CAR2 expression (Fig.3) was shown (Fig. 5 to 10) to contain putative Abf1p, Rap1p,and Dal82p protein binding sites. To determine whether eachof these putative binding sites participated in transcription, wecloned wild-type and mutant alleles of this fragment into the2mm-based expression vector pHP52. As shown in Fig. 11, thisplasmid supported high-level reporter gene expression but ex-hibited only a very modest response when OXLU was added tothe medium (plasmid pHP152). This modest induction was notsurprising because the CAR2 fragment had just been shown tocontain putative Abf1 and Rap1 protein binding sites (data inFig. 5 to 8). If functional Abf1p and Rap1p sites were present,they would participate in activating significant reporter geneexpression in the absence of OXLU, which in turn would resultin a relatively poor induction response due to the high level ofbasal expression. To test this possibility, we synthesized a DNAfragment that contained the putative Abf1p and Rap1p bind-ing sequences, cloned it into the expression vector pHP52 (toyield plasmid pHP150), and assayed the reporter gene expres-

sion it supported. As anticipated, plasmid pHP150 supportedhigh-level, OXLU-independent reporter gene expression (Fig.11). This expression, however, was only about one-half to one-fourth the level observed when the putative Dal82p bindingsite was also present (Fig. 11, compare plasmids pHP152 andpHP150). The slight decrease in activity observed when OXLUwas present is a common observation when OXLU is added tocultures; it is due to toxic effects of OXLU on the cell (8, 9). Toassess the contribution of the putative Abf1p binding site con-tained in plasmid pHP150, we mutated the putative Abf1p sitein the same way as we had in the EMSA experiments (Fig. 5and 6). Gene expression supported by the mutant DNA frag-ment (plasmid pHP163) was about half that supported by itswild-type counterpart (plasmid pHP150) and only about 10%of that observed with plasmid pHP152. This result demon-strates that a mutation that eliminates putative Abf1p binding(Fig. 5 and 6) also decreases reporter gene transcription hereand supports the idea that these CAR2 sequences comprise afunctional Abf1p binding site.

We next mutated the putative Rap1p binding site as we didfor the experiments whose results are shown in Fig. 7 and 8(Fig. 11, plasmid pHP164); this resulted in a seven- to ninefolddecrease in lacZ expression compared to that of the wild-typesequence (plasmid pHP150). With the aforementioned data,reporter gene expression supported by plasmid pHP150 can beconcluded to be largely due to the sequences homologous tothe Abf1p and Rap1p sites. Furthermore, the putative Rap1pbinding site appears to drive greater reporter gene expressionthan the putative Abf1p site.

We next focused on the putative Dal82p binding site situatedbetween CAR2 positions 2254 to 2235. As before, we synthe-

FIG. 9. Protein binding to CAR2 and DAL7 DNA fragments. DNA frag-ments labeled with 32P at their 59 ends and containing the JRD72X and RD43sequences indicated in Fig. 1 were used as probes. An extract (EXT) from IPTG(isopropyl-b-D-thiogalactopyranoside)-induced E. coli cells harboring plasmidpRD4 (T7) or pRD41 (T-7–DAL82) was prepared as described previously (17).EMSA reaction conditions were exactly as described in the work of Dorringtonand Cooper (17).

FIG. 10. Competition between CAR2 and DAL7 DNA fragments. An oligo-nucleotide labeled at its 59 end with 32P and covering DAL7 positions 2241 to2206 (Fig. 1, JRD72X) was used as the probe. The reaction mixtures in lanes Athrough E contained decreasing amounts of the unlabeled CAR2 wild-type oli-gonucleotide (Fig. 1, RD43), and lanes I through M contained increasingamounts of the unlabeled CAR2 mutant oligonucleotide (mut) (Fig. 1, RD44).The remaining conditions and procedures used in the experiment were as de-scribed in the legend to Fig. 6. COMP, competitor; EXT, extract; g, microgramquantities.

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sized an oligonucleotide containing these positions, cloned itinto an expression vector, and assayed its ability to supportreporter gene expression. The plasmid containing this oligo-nucleotide was unable to support b-galactosidase productionabove the background level (Fig. 11, plasmids pHP151 andpHP52). This was consistent with earlier data showing that theDAL7 UISALL (Dal82p binding site) is unable to support tran-scription in a heterologous expression vector in the absence ofa GATAA-containing UASNTR site (Gln3p and/or Gat1p bind-ing site) (9).

The observations that plasmid pHP150 supports only one-third of the lacZ expression of the parent plasmid pHP152 andthat the remainder of plasmid pHP152 (i.e., plasmid pHP151)supports none at all suggest that the putative Dal82p bindingsite may function synergistically with the putative Rap1p siteand/or Abf1p site (Fig. 11). Indeed, the full-length fragment(plasmid pHP152) supported significantly greater transcriptionthan the sum of the levels of transcription of the isolatedfragments (plasmids pHP150 and pHP151). To test this sug-gestion relative to the involvement of the Rap1p site, we syn-thesized an oligonucleotide containing only the putative Rap1pand Dal82p binding sites (positions 2269 to 2235) and ana-lyzed its ability to support transcription (Fig. 11, plasmidpHP184). This plasmid supported about two-thirds of the lacZexpression observed with plasmid pHP152 and five- to six-fold-greater expression than with plasmid pHP163. To evaluate thecontributions of the individual sites on plasmid pHP184, wefirst mutated the putative Rap1p site (plasmid pHP185). Thismutation decreased b-galactosidase production 30- to 40-foldto a level slightly above background. Mutation of the putativeDal82p binding site of plasmid pHP184 decreased the level ofreporter gene expression by about three-fourths. Moreover,the remaining gene expression supported by this plasmid wasabolished after addition of OXLU (Fig. 11, plasmid pHP186);this result was surprising and was not reproducible. As ex-pected, mutation of both the putative Rap1p and Dal82p bind-

ing sites completely destroyed reporter gene expression (Fig.11, plasmid pHP187). These data support the contention thatRap1p and Dal82p act synergistically to support reporter geneexpression.

Finally, we analyzed the phenotypes of the above-describedset of cis-acting mutations in the context of the entire fragmentfrom 2303 to 2235 (Fig. 12). Mutation of the putative Abf1pbinding site (plasmid pHP153) decreased reporter gene ex-pression by 10 to 20% compared to that of the parent (plasmidpHP152). In contrast, mutation of the putative Rap1p bindingsite (pHP154) decreased reporter gene expression 12- to 14-fold. In both instances, however, the presence of OXLU re-sulted in about 20% greater expression. Mutation of both theputative Rap1p and Abf1p sites together eliminated transcrip-tion (plasmid pHP156). These data corroborate those shown inFig. 11.

The contribution of a putative Dal82p binding site to overallreporter gene expression supported by plasmid pHP152 ap-pears unimpressive if it is evaluated on the basis of an OXLUresponse. However, when mutated, the putative Dal82p bind-ing site of plasmid pHP152 exhibits a more dramatic effect.This mutant site supported only 20 to 40% of the reporter geneexpression supported by plasmid pHP152 (Fig. 12). Moreover,addition of OXLU in this case did not result in an increase inreporter gene expression but instead resulted in a small de-crease. These results suggest that there is a putative Dal82pbinding site participating in lacZ expression supported by plas-mid pHP152.

Requirement of Dal82p for OXLU-mediated reporter geneexpression. OXLU-induced allantoin pathway gene expressionrequires participation of the Dal82 and Dal81 proteins (8, 9).Therefore, we determined whether these regulatory proteinswere required for reporter gene expression supported by plas-mid pHP152. b-Galactosidase production decreased by up to75% in the dal82 deletion mutant relative to that of the wildtype (Fig. 13, plasmid pHP152). Moreover, this decrease was

FIG. 11. b-Galactosidase production supported by plasmids containing synthetic CAR2 DNA fragments. The synthesized DNA fragments were cloned into theheterologous expression vector pHP52. Plasmids pHP150 and pHP184 contain native CAR2 sequences, whereas their derivatives contain mutated sequences in whichthe mutations are shown with lowercase letters. The transformation recipient was strain TCY1.

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independent of OXLU. However, no decrease in activity wasobserved in dal81 deletion mutant cells grown without an in-ducer and decreased only 25% when it was present (Fig. 13,plasmid pHP152). Mutation of the putative Dal82p bindingsite in plasmid pHP152 resulted in reporter gene expressionthat was the same as in wild-type, dal82, and dal81 strains (Fig.13, plasmid pHP155). Incidentally, this was also the level ob-served with plasmid pHP152 expressed in the dal82 deletion.We repeated the above experiments using vectors containingonly the putative Rap1p and Dal82p binding sites (Fig. 13,plasmids pHP184 and pHP186). Results similar to those ob-served with the entire DNA fragment (plasmid pHP152) wereobtained. Again, mutation of the Dal82p binding site or dele-tion of the DAL82 gene resulted in three- and fivefold de-creases in b-galactosidase production in proline medium in thepresence and absence of OXLU, respectively. In no case wasDal81p necessary for basal-level activity, indicating thatDal82p but not Dal81p is important for reporter gene expres-sion in the absence of OXLU.

An important test to determine if the three sites describedabove in fact function in CAR2 transcription is to evaluate the

effects of mutations in these sites in the context of a full-lengthCAR2 promoter. CAR2, as mentioned earlier, contains se-quences homologous to the URS1 site of CAR1, the Ume6p(also called Car80p) repressor binding site (1, 49–51). In ouranalysis of the CAR1 promoter, we found that the presence ofthis strong repressor binding site masks some of the effects ofmutations in the weaker UAS elements of that promoter (47).The contribution of these weak UASs could be unmasked bymutating the URS1 site. Therefore, we constructed full-lengthwild-type and mutant UAS alleles in both wild-type and urs1-minus promoter backgrounds (Fig. 13).

Mutation of the putative Abf1p binding site in the context ofa full-length CAR2 promoter did not bring about a demonstra-ble decrease in b-galactosidase production when the URS1 sitewas intact (Fig. 14, compare plasmids pHP201 and pHP203).In a urs1 mutant background, however, reporter gene expres-sion decreased about one-third when cultures were grown inproline or proline plus OXLU medium (compare plasmidspHP202 and pHP204). A similar decrease was not observedunder conditions of high-level arginine-mediated induction.Mutation of the putative Rap1p binding site, on the other

FIG. 12. b-Galactosidase production supported by plasmids containing synthetic DNA fragments covering the CAR2 regulatory region between positions 2303 and2235, which were cloned into the expression vector pHP52. The transformation recipient and conditions were as described for Fig. 11.

FIG. 13. b-Galactosidase production in wild-type (TCY1) and dal82 (RDY4) or dal81 (HEY6) deletion mutant strains supported by plasmids containing syntheticDNA fragments covering the CAR2 regulatory regions that were cloned into the expression vector pHP52. WT, wild type.

7060 PARK ET AL. J. BACTERIOL.

hand, produced about a threefold decrease regardless of themedium used and whether or not the URS1 site was mutated(compare plasmid pHP205 with plasmid pHP206). Mutation ofboth the putative Abf1p and Rap1p sites resulted in about a50% decrease relative to the situation when only the Rap1psite was mutated under all conditions tested (Fig. 14, comparepHP205 with pHP211 and pHP206 with pHP212). The doublemutant promoter retained only 10 to 20% of the wild-typeexpression levels regardless of the growth conditions or thepresence of a functional URS1 element. This result argues thateven though CAR2 is induced by arginine, a large part of itstranscription depends upon constitutively functioning Rap1p.A smaller contribution also appears to be made by a secondconstitutively functioning factor, Abf1p.

Finally, we assessed the contribution of the putative Dal82pbinding site to overall CAR2 expression in a similar manner.

Mutation of this site alone reduced b-galactosidase productionabout 30 to 50% when a wild-type URS1 allele was present(Fig. 15, compare plasmids pHP201 and pHP207). When theanalogous experiment was carried out with a urs1 allele, lacZexpression decreased 25 to 35% relative to wild-type expres-sion when proline was provided as the nitrogen source; nodifference was observed with arginine as the nitrogen source(Fig. 14, compare plasmid pHP202 with plasmid pHP208).

We next assayed the effects of mutating the putative Dal82pbinding site with and without the Abf1p and Rap1p sites. Thetriple mutant possessed only 15 to 30% of the wild-type ex-pression levels, and there was no effect whatever, beyond itsslight toxicity, when OXLU was added as the inducer. Further,there was only about one-fourth of the wild-type activity whenarginine was provided as the nitrogen source even though theelements necessary for arginine-mediated induction were un-

FIG. 14. Genetic analysis of the putative CAR2 Abf1p and Rap1p binding sites in the context of a full-length CAR2 promoter. CAR2 DNA fragments betweenpositions 2302 and 2132 containing wild-type or mutated putative CAR2 Abf1p and Rap1p binding sites were synthesized chemically or by PCR and cloned into SalIand BamHI sites of plasmid pHP200. Reporter gene expression supported by these plasmids was determined in cells grown in YNB medium containing proline (PRO),arginine (ARG), or proline plus OXLU (PRO1OXLU). Open and closed boxes in the inserts represent wild-type and mutated sites, respectively. T, TATA sequence.

FIG. 15. Combinational mutation analysis of the putative CAR2 Abf1p, Rap1p, and Dal82p binding sites in the context of a full-length CAR2 promoter. CAR2 DNAfragments between positions 2302 and 2132 containing wild-type or mutated putative CAR2 Abf1p, Rap1p, and Dal82p binding sites were synthesized chemically orby PCR and cloned into SalI and BamHI sites of plasmid pHP200. Growth, abbreviations, and other conditions were as described for Fig. 14.

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touched (Fig. 15, compare plasmid pHP201 with plasmidpHP213). The fold induction with arginine, however, remainedthe same as in the wild type, indicating the continued function-ing of the arginine-dependent transcription factors (the argi-nine value divided by the proline value). These data demon-strate the level of expression that can be supported by thearginine induction system alone. When the experiment wasrepeated with a plasmid in which the urs1 site was also mu-tated, a slightly smaller fraction of wild-type activity was re-tained (6 to 17%) (Fig. 15, compare plasmid pHP202 withplasmid pHP214). Here, as with plasmid pHP213, there was noOXLU-mediated induction and arginine-induced expressionreached only 20% of the wild-type level.

Another way to visualize the contribution of the putativeDal82p binding site is to compare the level of expression ob-served with mutations in the Abf1p and Rap1p sites with thatobserved when all three sites (Abf1p, Rap1p, and Dal82p)were mutated. Arginine-induced reporter gene expression inthe Abf1p and Rap1p site double mutant was almost twice asgreat as when the Dal82p site was also mutated in a URS1allele. There was no difference in the values observed with thedouble mutant when proline or proline plus OXLU was pro-vided (Fig. 15, compare pHP214 with pHP212 and pHP213with pHP211). When the experiment was performed with aurs1 allele, b-galactosidase production was up twofold in thedouble mutant under all conditions compared to that of thetriple mutant (Fig. 15, compare plasmids pHP212 andpHP214). Again, however, there was no OXLU-induced ex-pression. The lack of OXLU-induced expression in these ex-periments is not surprising given the necessity of Rap1p func-tion for the Dal82p site to mediate transcription. These dataargue that although the Dal82p site functions synergisticallywith the Rap1p site with respect to OXLU-induced induction,the Dal82p site also appears to mediate a twofold enhance-ment of arginine-induced expression.

DISCUSSION

Two criteria must be met to conclude that a promoter con-tains a given transcription factor binding site: (i) the transcrip-tion factor must be shown to bind to the putative site and (ii)the putative site must be shown to be required for or to par-ticipate in the gene’s expression. Qualitative EMSAs describedin this work fulfilled the first of these criteria by demonstratingthat Rap1p, Abf1p, and Dal82p bind to sites in the CAR2upstream region. Based on crude approximations derived fromthe competition EMSA data, the Rap1p site in CAR2 is areasonably strong one; the CAR2 fragment containing theRap1p site was an effective competitor of the TEF2 fragmentcontaining a strong, well-characterized Rap1p site for proteinbinding. The Dal82p binding site in CAR2 was also relativelystrong in that its ability to serve as a competitor comparedfavorably with that of an analogous site in a DAL7 fragment,one of the better binding sites found among the allantoinpathway genes. In contrast, Abf1p does not bind to the CAR2fragment as well as it does to the ARS1 fragment, which con-tains a well-characterized Abf1p site. The second criterion wasfulfilled through genetic analysis of CAR2 promoter fragmentscloned into a heterologous expression vector and full-lengthpromoters fused to lacZ. These results demonstrated that se-quences needed for Rap1p, Abf1p, and Dal82p binding arealso required for maximum levels of CAR2 transcription. To-gether, the genetic and biochemical results of this work permitus to conclude that the CAR2 promoter contains functionalRap1p, Abf1p, and Dal82p binding sites in addition to the

previously identified Ume6p (also called Car80p) and UASARGsites.

Our results support the conclusion that regulated CAR2expression depends upon transcriptional activation by consti-tutively operating global activators (Abf1p and Rap1p) beingbalanced by a similarly strong repression being exerted by aglobal repressor, Ume6p (also called Car80p) (51). The bal-ance of positive and negative control is then tipped towardexpression when an inducer is present and toward quiescencewhen it is not. In the case of CAR2, inducers exert their effectsthrough the ArgR proteins in the presence of arginine andDal81p and Dal82p when OXLU is provided. This formalmodel of transcriptional regulation is identical to that firstproposed for CAR1 (47, 51). It also explains the early obser-vation that ornithine transaminase production is increasedwith urea as the nitrogen source (23); urea is the precursor ofallophanate, the naturally occurring analogue of OXLU (9).

This work also extends our understanding of the Dal81 andDal82 proteins. Prior to our experiments, functional UISALLsites had been found only upstream of the allantoin pathwaygenes and were thought to operate exclusively in conjunctionwith GATA-containing UASNTR elements (9, 17, 57, 61). Inthis work we demonstrate that UISALL and associated Dal82pcan function in association with elements and factors otherthan the GATA-containing UASNTR elements and their asso-ciated Gln3p and/or Gat1p transcriptional activator. This con-clusion is supported by the facts that the CAR2 promoter isdevoid of UASNTR elements and that the role(s) played byGln3p and/or Gat1p for the allantoin pathway genes is pre-dominantly provided by Rap1p in the case of CAR2.

The suggestion that Dal82p mediates CAR2 transcription viasynergistic association with Rap1p influences the interpreta-tion of earlier experiments concerning Dal82p and Gln3p (57).These experiments showed that placing a functional Dal82pbinding site adjacent to a mutant uasNTR site, i.e., one con-taining a single mutation in the GATA sequence, suppressedthe mutant phenotype. Suppression required only a properlysituated UISALL element, functional Dal82p, and was not de-pendent upon Dal81p or an inducer. At the time these sup-pression data were reported, we suggested that Dal82p boundthe UISALL situated adjacent to the mutant uasNTR site, whichfacilitated or stabilized Gln3p binding to the latter site, poten-tially through a protein-protein interaction. This scenario re-mains a viable interpretation of those earlier results. However,the present data demonstrate that Dal82p can also functionsynergistically with a global transcription factor (Rap1p) hav-ing no specific relationship with nitrogen catabolism. Such aresult does not particularly support proposing a direct Gln3p-Dal82p interaction to account for the suppression data. Analternative possibility is that interactions mediating Dal82p-dependent stabilization of Gln3p binding may occur more in-directly, perhaps through involvement of components of theSAGA or related core transcription complexes.

Finally, the present work offers some insight into the bio-chemical characteristics of Dal82p operation. The suppressionexperiments just discussed are consistent with the idea thatUISALL and Dal82p are able to suppress the GATA mutationsin the absence of an inducer or Dal81p (57). In correlation withthese observations is the repeated demonstration in this workthat Dal82p can enhance transcription in association withRap1p or the transcription factors responsible for arginine-mediated induction, again in the absence of Dal81p or aninducer. In other words, the participation of Dal81p is notessential for all Dal82p functions and neither is an inducer. Onthe other hand, Dal81p is absolutely required for OXLU-induced gene expression. The separation of Dal81p and Dal82

7062 PARK ET AL. J. BACTERIOL.

functions observed here and earlier will significantly influencemechanistic studies aimed at understanding the biochemicalfunctions of these two transcription factors.

ACKNOWLEDGMENTS

We thank the members of the U.T. Yeast Group who read thismanuscript and offered suggestions for its improvement. Oligonucle-otides used in this study were prepared by the U.T. Molecular Re-source Center.

This work was supported by Public Health Service grant GM-35642from the National Institute of General Medical Sciences and the Ac-ademic Research Fund (GE-96-14) of the Ministry of Education, Re-public of Korea.

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