INTRODUCTION

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All nitrogen catabolite repression (NCR)-sensitive genes in Saccharomyces cerevisiae have GATA sequences in their promoters, and their expression is dependent upon one or both of the GATA family transcriptional activators Gln3p and/or Gat1p (also called Nil1p) (for a brief review of the GATA factor field, see the introductions of references 7 and 52). Gln3p has been shown to bind to the GATA sequences, and both Gln3p and Gat1p have the ability to activate transcription of 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 and Gat1p working synergistically with pathway-specific transcription factors (47, 61). Arginine and allantoin pathway gene expression is also subject to induction in response to the presence of small signal metabolites (8, 9, 23, 37-40, 59, 60). In the case of the arginine pathway genes (CAR genes), the inducer is arginine (59) and the transcription factors involved are ArgRIp, ArgRIIp, ArgRIIIp, and Mcm1p. For the allantoin pathway genes (DAL and DUR genes), the inducer is allophanate or its nonmetabolized analogue, oxalurate (OXLU), and the associated transcription factors are Dal82p and Dal81p (8, 9).

The CAR1 promoter has been extensively studied both biochemically and genetically (2-4, 11, 12, 14, 18, 23, 25, 59). It consists 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 Abf1 sites and a GC-rich sequence are necessary to achieve highly induced transcription levels of CAR1 (27-29, 47). The action of these strong, constitutively acting transcription factors is antagonized by the equally strong negative action of Ume6p (also called Car80p) bound to the URS1 site (1, 3, 42, 47-51). The metabolite-responsive promoter elements are much weaker than those just cited (47) and have been suggested to tip the balance either toward expression when the inducer is present and a repressing nitrogen source is absent or toward quiescence when arginine is absent and/or a repressive nitrogen source 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 induction (10, 11, 13, 14, 18, 19, 34-36). NCR-sensitive CAR1 expression, on the other hand, is mediated by two GATA sequences associated with Gln3p and Gat1p (47).

Although arginase and ornithine transaminase (encoded by CAR1 and CAR2) catalyze contiguous enzyme reactions in the arginine-degradative pathway of S. cerevisiae, production of the two enzymes is regulated somewhat differently (37-40, 59, 60). While arginine induces production of both arginase and ornithine transaminase, only arginase production is NCR sensitive (14, 37-40, 59, 60). Production of ornithine transaminase, on the other hand, is induced by allophanate, the last intermediate of the allantoin-degradative pathway, while CAR1 expression is not (23). Unlike that of CAR1, the CAR2 promoter has not been characterized.

The allantoin pathway gene promoters appear to be simpler than those in the arginine pathway (8, 9). For example, the DAL7 promoter contains three types of sites, each of which is associated with a different type of regulation. Multiple GATA sequences mediate NCR-sensitive gene expression that depends upon Gln3p and Gat1p (7-9, 51). DAL7 expression is down regulated by the action of Dal80p, which antagonizes transcriptional activation, mediated by Gln3p and Gat1p (9). Finally, inducer-dependent (allophanate or its nonmetabolized analogue OXLU) expression depends upon the UIS_ALL site; Dal82p binds to this site and Dal81p functions in association with it (8, 9, 17). A notable similarity exists in the formal organizations of the CAR and DAL promoters; the action of transcriptional activators is antagonized by repressor proteins, and the balance is shifted by the inducer-responsive transcription factor (11a, 47).

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

This work has three objectives, namely, to (i) identify the promoter elements of CAR2 responsible for allophanate-induced expression; (ii) determine whether the overall structure and operation of CAR2 is analogous to those features of CAR1, i.e., whether CAR2 contains strong, opposing positive and negative control elements, with the metabolite-responsive elements shifting the balance; and (iii) determine whether Dal82p can function in association with global transcription factors other than Gln3p and/or Gat1p, and if so, which ones. This work does not address elements and proteins associated with arginine-dependent transcriptional activation. This aspect of CAR2 gene regulation has been reported by Messenguy and Dubois and their colleagues.

	MATERIALS AND METHODS

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Strains and media. The S. cerevisiae strain used for -galactosidase assays was TCY1 (MAT lys2 ura3), and the wild-type strain used for electrophoretic mobility shift assays (EMSAs) was W303-1A (MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1). Strains HEY6 and RDY4 are isogenic derivatives of strain TCY1 containing deletions of the DAL81 and DAL82 genes, respectively. Strain YLS91 (29) (kindly provided by David Shore) is a derivative of W303-1A containing two RAP1 genes; one gene is disrupted by LEU2 (which results in a deletion between the two BamHI sites at nucleotides 818 and ca. 2200), and the other has nucleotides 894 to 1584 deleted, which results in a deletion of amino acids 44 to 303. The RAP1 DNA binding domain is located between residues 361 and 596 of the 827-amino-acid protein (24). Therefore, the truncated protein still retains the DNA binding sites (29). The Escherichia coli strain used for cloning was HB101 (supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1). Yeast cultures used for Northern blot analysis and -galactosidase assays were grown in medium containing 0.17% yeast nitrogen base without amino acids or ammonium sulfate (YNB medium) and supplemented with 2% glucose and 0.1% arginine (induced condition) or 0.1% proline (noninduced condition). OXLU was added to YNB-proline medium at a final concentration of 61 mg/ml for the CAR2 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 were grown to the mid-log phase (40 to 60 Klett units). Total RNA was isolated by the method of Carlson and Botstein (5). Poly(A)⁺ RNA was isolated with oligo(dT)-cellulose (Pharmacia Amersham) columns and resolved on 1.4% agarose-formaldehyde gels (49). The resolved RNAs were transferred to GeneScreen Plus nylon 66 (Dupont, NEN Research Products) membranes. The membranes were then hybridized to double-stranded DNA probes made radioactive by the randomly primed labeling method (Boehringer Mannheim). The procedures followed for hybridization and washing were those described in the GeneScreen Plus protocol manual.

PCR. PCR was carried out with BamHI-digested plasmid pRS427 containing the 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 5' regulatory region shown in each construct. The PCR mixture consisted of 0.5 µg of template DNA, 100 pmol of each primer, 2.5 U of Taq DNA polymerase, 0.25 mM concentrations 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 amplification consisted of one cycle of 94°C (5 min) and 30 cycles of 94°C (1 min), 37°C (1 min), and 72°C (3 min) followed by one cycle of 72°C (5 min). The PCR products were purified with Sephadex G25 spun columns and subjected to enzyme digestion.

Plasmid constructions. Plasmid constructions for the CAR2 5' deletion analysis were carried out by PCR. PCR products contained CAR2 DNA sequences between various 5' upstream positions (634, 530, 470, 420, 380, 350, 302, 251, 233, 205, 177, 165, and 120) and position +3 as well as SalI and BamHI sites at the 5' and 3' termini of the products, respectively. The fragments were digested with SalI and BamHI and were cloned into SalI and BamHI sites of plasmid pLG669Z, described in detail by Guarente et al. (20-22).

EMSA. The sequences of synthetic oligonucleotides used as DNA probes and competitors 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. The protocols 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 the DNA probes.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   Oligonucleotides used as radioactive probes and competitors in EMSAs. Sequences homologous to Abf1p, Rap1p, and Dal82p binding sites are indicated with brackets. Mutations in each site, here and in all other figures, are shown as lowercase letters. Numbers in the figure indicate the positions of the bases whose coordinates are given. mt, mutant.

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

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

	RESULTS

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Induced CAR2 expression. We first established the CAR2 baseline induction pattern using Northern blot analysis. Steady-state levels of CAR1 mRNA (our positive control gene) were much higher in cells provided with arginine as the nitrogen source than with proline, i.e., CAR1 expression was highly inducible. (Fig. 2, lanes A and B). Compared to those of CAR1, CAR2 mRNA levels are less responsive to arginine largely due to the fact that substantial basal-level CAR2 expression occurs when proline is provided as the sole nitrogen source (lanes C and E). CAR2 expression was induced by OXLU but to a much smaller degree than when arginine was used as the inducer (Fig. 2, lanes C and D).

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Steady-state levels of CAR1 and CAR2 mRNAs in S. cerevisiae grown on various nitrogen sources. Poly(A)+ RNA was prepared from S. cerevisiae 1278b cells grown in YNB media containing proline (PRO) or arginine (ARG). OXLU, a gratuitous inducer, was added to YNB-proline medium (PRO+OXLU) at a final concentration of 61 mg/ml for the CAR2 induction. The same designations for growth conditions are used throughout this work. Histone H4 mRNA served as a control for Northern blot loading and transfer efficiencies.

5' deletion analysis of the CAR2 upstream region. We began dissection of the CAR2 promoter by constructing a CAR2-lacZ fusion plasmid (pHP120) (see Materials and Methods) containing 634 bp of the upstream region. This plasmid supported two- and eightfold-higher levels of -galactosidase production in response to the presence of OXLU and arginine, respectively (Fig. 3). A series of deletions removing nucleotides 634 to 302 (plasmids pHP120 to pHP126) had little effect on -galactosidase production, suggesting that this region was not demonstrably required for regulated CAR2 expression (Fig. 3). Deletion of nucleotides 302 to 251 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 of the next 18 nucleotides to position 233 resulted in further 10-, 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 205 (plasmid pHP129) resulted in the loss of the remaining arginine-dependent -galactosidase production. The same result was observed with the three subsequent deletions, the last of which (plasmid pHP132) lacked all sequences 5' of position 120; the TATA sequence of CAR2 is situated at position 134. These data demonstrate that the region between CAR2 positions 302 and 233 contains elements that are important for high-level expression when arginine is used as the nitrogen source as well as for the more modest level of expression when OXLU is present.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   5' deletion analysis of the CAR2 upstream region. DNA fragments containing the CAR2 regulatory region as well as SalI and BamHI restriction sites at the 5' and 3' termini, respectively, were amplified from CAR2 DNA by PCR. The PCR products were digested with SalI and BamHI and cloned into the SalI and BamHI sites 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 represent sequences that are homologous to various transcription factor binding sites. T's indicate the positions of TATA sequences. Numbers at the left of each insert indicate the 5' 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 this and all subsequent figures.

Demonstration of a putative Abf1p binding to the region from 302 to 233 of CAR2. Inspection of the CAR2 upstream region from 302 to 233 revealed sequences homologous to ABF1, RAP1, UIS_ALL and UAS_ARG sites (Fig. 4). Notably absent were GATAA-containing sequences homologous to UAS_NTR. A priori, the presence of sequences homologous to known transcription factor binding sites is not sufficient reason to conclude that transcription factors bind to them and mediate CAR2 transcription. Therefore, EMSAs were performed to ascertain whether CAR2 promoter fragments containing these homologous sequences were indeed able to bind protein. When radioactively labeled CAR2 fragment HD189 (positions 313 to 271) was incubated with a wild-type yeast crude cell extract (see Materials and Methods), two retarded species were observed (Fig. 5, lane B); no retarded species was observed when extract was omitted from the EMSA reaction mixture (lane A). To assess the binding specificity, we used a mutant allele of fragment HD189 in which sequences homologous to an ABF1 binding site were altered in a way previously shown to destroy Abf1p binding (15, 16, 28). The slower-migrating DNA-protein complex was lost when the mutant promoter fragment was used as the probe (Fig. 5, lane C). These data suggested that only this slower-migrating species was potentially associated with Abf1p binding. The identity of the protein(s) in the more rapidly migrating complex in lanes B and C remains unknown.

View larger version (45K): [in this window] [in a new window]   FIG. 4.   CAR2 sequences homologous to the binding sites for Abf1p, Rap1p, and Dal82p.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Protein binding to ARS1 and CAR2 DNA fragments. DNA fragments labeled with 32P at their 5' ends and containing the HD189 and ARS1 sequences indicated in Fig. 1 were used as probes. Yeast cell extract (strain W303-1A) was omitted from the reaction mixture resolved in lanes A and D. All reaction mixtures contained a 200-fold excess of sheared calf thymus DNA by sonication as a nonspecific competitor. EXT, minus extract, W.T., wild type; mut, mutant.

View larger version (52K): [in this window] [in a new window]   FIG. 6.   Competition between CAR2 and ARS1 DNA fragments. An oligonucleotide covering CAR2 positions 313 to 271 (left) or ARS1 positions 748 to 780 and flanked by SalI and EagI restriction sites (right) was labeled at its 5' end with 32P and used as the probe. Competitor DNA was omitted in the reaction mixtures resolved in 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 (). The remaining conditions and procedures used in the experiment were as described in the legend to Fig. 5. Strain W303-1A was the source of the crude cell extract (EXT).

Demonstration of putative Rap1p binding to the region from 302 to 233 of CAR2. We next investigated a CAR2 sequence contained in DNA fragment HD190 (CAR2 positions 282 to 247) which was homologous to known Rap1p DNA binding sites (Fig. 4). One major and one minor retarded species were observed in the EMSA when extract from the wild-type strain W303-1A was used as the source of protein (Fig. 7, lane B). When the assay was repeated with an extract derived from the rap1 mutant strain YLS91 (carrying a truncated, but binding-competent form of Rap1 [6, 24, 29, 30, 46]), the major retarded species migrated to a new location just below the minor species observed in lane B (Fig. 7, lane C). When a mutant CAR2 DNA probe (fragment HD192, in which the Rap1p site-homologous sequence is mutated) was used in the assay, little if any of the more slowly migrating DNA-protein complex present in lane B was observed (Fig. 7, lane D). The more rapidly migrating species in lane B was also observed in lane D, arguing that it was not associated with the mutated sequence in fragment HD192. The small amount of DNA-protein complex in lanes C and D that possessed the same mobility as the major complex observed in lane B is unlikely to be Rap1p specific because it was also observed in lane C, which did not contain full-length Rap1p. We compared the data shown in Fig. 7, lanes A to D, with that obtained with a similarly sized, well-characterized TEF2 DNA probe previously 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 the CAR2 fragment was used (lanes A to D). The EMSA using the TEF2 probe also exhibited the nonspecific protein-DNA complexes seen in lanes B to D. 

View larger version (41K): [in this window] [in a new window]   FIG. 7.   Wild-type and truncated Rap1p binding to CAR2 and TEF2 DNA fragments. DNA fragments 32P labeled at their 5' ends and containing the HD190 and HD192 sequences indicated in Fig. 1 were used as probes. Yeast cell extract (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 in lanes C and G (rap1).

View larger version (66K): [in this window] [in a new window]   FIG. 8.   Competition between CAR2 and TEF2 DNA fragments for protein binding. An oligonucleotide 32P labeled at its 5' end and covering CAR2 positions 282 to 247 (left) or TEF2 positions 449 to 414 (right) was used as the probe. The reaction mixtures in lanes A through F contained decreasing amounts of the unlabeled TEF2 (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 TEF2 mutant (mut.) (left) or CAR2 mutant (right) oligonucleotide. The remaining conditions and procedures used in the experiment were as described in the legend to Fig. 6. EXT, extract; , microgram amounts.

Demonstration of putative Dal82p binding to the region from 302 to 233 of CAR2. The last CAR2 sequence we investigated was one homologous to UIS_ALL elements that are situated upstream of allantoin pathway genes (Fig. 4). These elements are Dal82p binding sites that mediate induction of DAL and DUR gene expression when allophanate or its analogue, OXLU, is present (9, 17). To determine whether Dal82p bound to the CAR2 UIS_ALL-homologous sequence (272 to 211), CAR2 fragment RD43 (Fig. 1) was incubated with extracts derived from an E. coli culture transformed with a T-7 expression vector plasmid (pRD4) or one containing a T-7-DAL82 fusion (plasmid pRD41) (Fig. 9, lanes D to F). No signal was observed with extract derived from a transformant containing plasmid pRD4 alone (lane E). In contrast, several DNA-protein complexes were observed with extract derived from cells containing the plasmid bearing the T-7-DAL82 fusion (Fig. 9, lane F). Two of these complexes (indicated by arrows) were specific to the presence of Dal82p. The Dal82p-specific signal with the fastest mobility has been previously shown to derive from a Dal82p degradation product (17). The source of the weak nonspecific signal observed in the lowest-mobility complex is unknown (Fig. 9, lanes E and F). DAL7 DNA fragment RD72X, which contains a well-characterized DAL UIS_ALL element (17), was used in a control experiment to identify the relative mobility of a bona fide UIS_ALL-Dal82p complex (Fig. 9, lanes A to C). In this experiment, the major complexes with the lowest and greatest mobilities were the only ones that were Dal82p specific and migrated to positions similar to those observed in lanes D to F. 

View larger version (30K): [in this window] [in a new window]   FIG. 9.   Protein binding to CAR2 and DAL7 DNA fragments. DNA fragments labeled with 32P at their 5' ends and containing the JRD72X and RD43 sequences indicated in Fig. 1 were used as probes. An extract (EXT) from IPTG (isopropyl--D-thiogalactopyranoside)-induced E. coli cells harboring plasmid pRD4 (T7) or pRD41 (T-7-DAL82) was prepared as described previously (17). EMSA reaction conditions were exactly as described in the work of Dorrington and Cooper (17).

View larger version (76K): [in this window] [in a new window]   FIG. 10.   Competition between CAR2 and DAL7 DNA fragments. An oligonucleotide labeled at its 5' end with 32P and covering DAL7 positions 241 to 206 (Fig. 1, JRD72X) was used as the probe. The reaction mixtures in lanes A through E contained decreasing amounts of the unlabeled CAR2 wild-type oligonucleotide (Fig. 1, RD43), and lanes I through M contained increasing amounts of the unlabeled CAR2 mutant oligonucleotide (mut) (Fig. 1, RD44). The remaining conditions and procedures used in the experiment were as described in the legend to Fig. 6. COMP, competitor; EXT, extract; , microgram quantities.

Participation of the putative Abf1p, Rap1p, and Dal82p binding sites in transcription. The 70-bp CAR2 fragment (303 to 235), 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 each of these putative binding sites participated in transcription, we cloned wild-type and mutant alleles of this fragment into the 2µm-based expression vector pHP52. As shown in Fig. 11, this plasmid supported high-level reporter gene expression but exhibited only a very modest response when OXLU was added to the medium (plasmid pHP152). This modest induction was not surprising because the CAR2 fragment had just been shown to contain putative Abf1 and Rap1 protein binding sites (data in Fig. 5 to 8). If functional Abf1p and Rap1p sites were present, they would participate in activating significant reporter gene expression in the absence of OXLU, which in turn would result in a relatively poor induction response due to the high level of basal expression. To test this possibility, we synthesized a DNA fragment that contained the putative Abf1p and Rap1p binding sequences, cloned it into the expression vector pHP52 (to yield plasmid pHP150), and assayed the reporter gene expression it supported. As anticipated, plasmid pHP150 supported high-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 binding site was also present (Fig. 11, compare plasmids pHP152 and pHP150). The slight decrease in activity observed when OXLU was present is a common observation when OXLU is added to cultures; it is due to toxic effects of OXLU on the cell (8, 9). To assess the contribution of the putative Abf1p binding site contained in plasmid pHP150, we mutated the putative Abf1p site in the same way as we had in the EMSA experiments (Fig. 5 and 6). Gene expression supported by the mutant DNA fragment (plasmid pHP163) was about half that supported by its wild-type counterpart (plasmid pHP150) and only about 10% of that observed with plasmid pHP152. This result demonstrates that a mutation that eliminates putative Abf1p binding (Fig. 5 and 6) also decreases reporter gene transcription here and supports the idea that these CAR2 sequences comprise a functional Abf1p binding site.

View larger version (26K): [in this window] [in a new window]   FIG. 11.   -Galactosidase production supported by plasmids containing synthetic CAR2 DNA fragments. The synthesized DNA fragments were cloned into the heterologous expression vector pHP52. Plasmids pHP150 and pHP184 contain native CAR2 sequences, whereas their derivatives contain mutated sequences in which the mutations are shown with lowercase letters. The transformation recipient was strain TCY1.

View larger version (21K): [in this window] [in a new window]   FIG. 12.   -Galactosidase production supported by plasmids containing synthetic DNA fragments covering the CAR2 regulatory region between positions 303 and 235, which were cloned into the expression vector pHP52. The transformation recipient and conditions were as described for Fig. 11.

Requirement of Dal82p for OXLU-mediated reporter gene expression. OXLU-induced allantoin pathway gene expression requires participation of the Dal82 and Dal81 proteins (8, 9). Therefore, we determined whether these regulatory proteins were required for reporter gene expression supported by plasmid pHP152. -Galactosidase production decreased by up to 75% in the dal82 deletion mutant relative to that of the wild type (Fig. 13, plasmid pHP152). Moreover, this decrease was independent of OXLU. However, no decrease in activity was observed in dal81 deletion mutant cells grown without an inducer and decreased only 25% when it was present (Fig. 13, plasmid pHP152). Mutation of the putative Dal82p binding site in plasmid pHP152 resulted in reporter gene expression that was the same as in wild-type, dal82, and dal81 strains (Fig. 13, plasmid pHP155). Incidentally, this was also the level observed with plasmid pHP152 expressed in the dal82 deletion. We repeated the above experiments using vectors containing only the putative Rap1p and Dal82p binding sites (Fig. 13, plasmids pHP184 and pHP186). Results similar to those observed with the entire DNA fragment (plasmid pHP152) were obtained. Again, mutation of the Dal82p binding site or deletion of the DAL82 gene resulted in three- and fivefold decreases in -galactosidase production in proline medium in the presence and absence of OXLU, respectively. In no case was Dal81p necessary for basal-level activity, indicating that Dal82p but not Dal81p is important for reporter gene expression in the absence of OXLU.

View larger version (16K): [in this window] [in a new window]   FIG. 13.   -Galactosidase production in wild-type (TCY1) and dal82 (RDY4) or dal81 (HEY6) deletion mutant strains supported by plasmids containing synthetic DNA fragments covering the CAR2 regulatory regions that were cloned into the expression vector pHP52. WT, wild type.

View larger version (17K): [in this window] [in a new window]   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 between positions 302 and 132 containing wild-type or mutated putative CAR2 Abf1p and Rap1p binding sites were synthesized chemically or by PCR and cloned into SalI and 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 (PRO+OXLU). Open and closed boxes in the inserts represent wild-type and mutated sites, respectively. T, TATA sequence.

View larger version (17K): [in this window] [in a new window]   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 DNA fragments between positions 302 and 132 containing wild-type or mutated putative CAR2 Abf1p, Rap1p, and Dal82p binding sites were synthesized chemically or by PCR and cloned into SalI and BamHI sites of plasmid pHP200. Growth, abbreviations, and other conditions were as described for Fig. 14.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Two criteria must be met to conclude that a promoter contains a given transcription factor binding site: (i) the transcription factor must be shown to bind to the putative site and (ii) the putative site must be shown to be required for or to participate in the gene's expression. Qualitative EMSAs described in this work fulfilled the first of these criteria by demonstrating that Rap1p, Abf1p, and Dal82p bind to sites in the CAR2 upstream region. Based on crude approximations derived from the competition EMSA data, the Rap1p site in CAR2 is a reasonably strong one; the CAR2 fragment containing the Rap1p site was an effective competitor of the TEF2 fragment containing a strong, well-characterized Rap1p site for protein binding. The Dal82p binding site in CAR2 was also relatively strong in that its ability to serve as a competitor compared favorably with that of an analogous site in a DAL7 fragment, one of the better binding sites found among the allantoin pathway genes. In contrast, Abf1p does not bind to the CAR2 fragment as well as it does to the ARS1 fragment, which contains a well-characterized Abf1p site. The second criterion was fulfilled through genetic analysis of CAR2 promoter fragments cloned into a heterologous expression vector and full-length promoters fused to lacZ. These results demonstrated that sequences needed for Rap1p, Abf1p, and Dal82p binding are also required for maximum levels of CAR2 transcription. Together, the genetic and biochemical results of this work permit us to conclude that the CAR2 promoter contains functional Rap1p, Abf1p, and Dal82p binding sites in addition to the previously identified Ume6p (also called Car80p) and UAS_ARG sites.

Our results support the conclusion that regulated CAR2 expression depends upon transcriptional activation by constitutively operating global activators (Abf1p and Rap1p) being balanced by a similarly strong repression being exerted by a global repressor, Ume6p (also called Car80p) (51). The balance of positive and negative control is then tipped toward expression when an inducer is present and toward quiescence when it is not. In the case of CAR2, inducers exert their effects through the ArgR proteins in the presence of arginine and Dal81p and Dal82p when OXLU is provided. This formal model of transcriptional regulation is identical to that first proposed for CAR1 (47, 51). It also explains the early observation that ornithine transaminase production is increased with urea as the nitrogen source (23); urea is the precursor of allophanate, the naturally occurring analogue of OXLU (9).

This work also extends our understanding of the Dal81 and Dal82 proteins. Prior to our experiments, functional UIS_ALL sites had been found only upstream of the allantoin pathway genes and were thought to operate exclusively in conjunction with GATA-containing UAS_NTR elements (9, 17, 57, 61). In this work we demonstrate that UIS_ALL and associated Dal82p can function in association with elements and factors other than the GATA-containing UAS_NTR elements and their associated Gln3p and/or Gat1p transcriptional activator. This conclusion is supported by the facts that the CAR2 promoter is devoid of UAS_NTR elements and that the role(s) played by Gln3p and/or Gat1p for the allantoin pathway genes is predominantly provided by Rap1p in the case of CAR2.

The suggestion that Dal82p mediates CAR2 transcription via synergistic association with Rap1p influences the interpretation of earlier experiments concerning Dal82p and Gln3p (57). These experiments showed that placing a functional Dal82p binding site adjacent to a mutant uas_NTR site, i.e., one containing a single mutation in the GATA sequence, suppressed the mutant phenotype. Suppression required only a properly situated UIS_ALL element, functional Dal82p, and was not dependent upon Dal81p or an inducer. At the time these suppression data were reported, we suggested that Dal82p bound the UIS_ALL situated adjacent to the mutant uas_NTR site, which facilitated or stabilized Gln3p binding to the latter site, potentially through a protein-protein interaction. This scenario remains a viable interpretation of those earlier results. However, the present data demonstrate that Dal82p can also function synergistically with a global transcription factor (Rap1p) having no specific relationship with nitrogen catabolism. Such a result does not particularly support proposing a direct Gln3p-Dal82p interaction to account for the suppression data. An alternative possibility is that interactions mediating Dal82p-dependent stabilization of Gln3p binding may occur more indirectly, perhaps through involvement of components of the SAGA or related core transcription complexes.

Finally, the present work offers some insight into the biochemical characteristics of Dal82p operation. The suppression experiments just discussed are consistent with the idea that UIS_ALL and Dal82p are able to suppress the GATA mutations in the absence of an inducer or Dal81p (57). In correlation with these observations is the repeated demonstration in this work that Dal82p can enhance transcription in association with Rap1p or the transcription factors responsible for arginine-mediated induction, again in the absence of Dal81p or an inducer. In other words, the participation of Dal81p is not essential for all Dal82p functions and neither is an inducer. On the other hand, Dal81p is absolutely required for OXLU-induced gene expression. The separation of Dal81p and Dal82 functions observed here and earlier will significantly influence mechanistic studies aimed at understanding the biochemical functions of these two transcription factors.