Vol. 174, No. 6

JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 1828-1836

0021-9193/92/061828-09$02.00/0 Copyright X 1992, American Society for Microbiology

Sequence of the GLNJ Gene of Saccharomyces cerevisiae: Role of the Upstream Region in Regulation of Glutamine Synthetase Expression PATRICIA L. MINEHART AND BORIS MAGASANIK* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307 Received 10 September 1991/Accepted 6 January 1992

The GLN1 gene, encoding glutamine synthetase in Saccharomyces cerevisiae, was sequenced, and its encoded polypeptide was shown to have significant homology to other eukaryotic glutamine synthetases. S1 analysis has defined the transcriptional start site of the gene. Upstream analysis of the gene using lacZ fusions has verified transcriptional control of the gene and has identified a nitrogen upstream activation sequence which is required for the increased transcription of GLNI seen when glutamine is replaced by glutamate as the nitrogen source. cis-acting sites required for the increased transcription in response to purine starvation also have been localized.

Fragments were purified from agarose by electroelution. Some ligations were performed in SeaPlaque low-melting agarose (FMC BioProducts, Rockland, Maine) as previously described (7). Escherichia coli plasmid minipreparations used in initial screenings of putative constructs were performed as described previously (14). For sequencing of the GLN1 gene, double-stranded DNA templates were prepared by purification over a CsCl gradient (17). For sequencing to check other constructs, Qiagen minipreparations (QIAGEN, Inc., Studio City, Calif.) were used as DNA templates. Yeast plasmid preparations were as described previously (32). Construction of the GLN14lacZ fusions. For construction of the larger GLNl-lacZ fusion plasmid, pSLFA178K, an upstream activation sequence (UAS)-lacking CYCI-lacZ fusion vector (10), was first treated with the restriction enzyme BamHI and then with the large fragment of DNA polymerase I to render the ends flush. The BamHI site of pSLFA178K lies within the lacZ gene, just downstream of the ATG. The vector was then treated with the restriction enzyme XhoI, which, with respect to the lacZ gene, cuts upstream from the BamHI site. The resulting vector was now missing the CYCl UAS sequences and the first three amino acids of ,B-galactosidase. A 705-bp SalI-XbaI GLNI fragment was isolated from plasmid pTK3, a Bluescript KS' vector in which the GLNJ SalI-XbaI fragment had been cloned. pTK3 was treated first with the restriction enzyme XbaI and second with the large fragment of DNA polymerase I to render the ends flush. The GLNI insert was then released upon treatment with XhoI, whose recognition site is adjacent to the Sall recognition site in the Bluescript vector. This fragment was then ligated into the pSLFA178K vector. The resulting plasmid contains 659 bp upstream from the GLN1 translational start site and the first 16 amino acids of GLNI; lacZ is fused in frame at amino acid residue 17, which contains one nucleotide from GLNI and two nucleotides from lacZ. For the smaller GLNI-lacZ fusion, pSLFA178K was first treated with the restriction enzymes BamHI and Asp 718, which cuts upstream from BamHI, and then with the large fragment of DNA polymerase I to render the ends flush. Plasmid pTK3 was treated with the restriction enzymes AvaIl and XbaI to release the 597-bp GLNI fragment and with the large fragment of DNA polymerase I to produce blunt ends. This fragment was then ligated into the pSLFA178K vector, and

Glutamine synthetase catalyzes the condensation of ammonia with glutamate to yield glutamine at the expense of an adenosine 5'-triphosphate phosphodiester bond. Since it catalyzes one of only two known reactions in which ammonia is used as a substrate, it plays an important role in the assimilation of inorganic nitrogen. Glutamine is not only directly incorporated into protein, but it is also the nitrogen donor for several reactions that result in the synthesis of other amino acids, nucleotides, and polysaccharides. In Saccharomyces cerevisiae, glutamine synthetase is uniquely encoded by the GLNJ gene. Regulation of GLNI is controlled by three separate systems which reflect different physiological roles of glutamine synthetase (1, 24). The first and most pronounced regulatory system increases transcription of GLNJ upon a shift from glutamine to glutamate as the nitrogen source. This increase in transcription with glutamate is dependent on the positively acting GLN3 gene product. The second regulatory system which controls GLNI transcription responds to purine limitation, while the third system responds to amino acid starvation via the general amino acid control pathway. Here we report the sequencing of the GLNJ gene and analysis of its upstream region to determine which sites are important for the control of the gene. We show that GLNI sites involved in the nitrogen control response are separate and upstream from the sites involved in purine regulation. We also demonstrate that GLNJ has a transcriptional start site which does not change upon activation by the various control systems. MATERUILS AND METHODS Strains and media. The S. cerevisiae strains used in these studies are listed in Table 1 and are isogenic to the wild-type strain PM38. This strain is three-quarters S288C background (American) and one-quarter 11278 background (Belgian). All media were made as described previously (23, 30). DNA manipulation and preparation. DNA manipulations were done by the method of Maniatis et al. (17), and enzymes were used according to the recommendations of the suppliers (New England BioLabs, Inc., Beverly, Mass., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.). *

Corresponding author. 1828

VOL. 174, 1992

TRANSCRIPTIONAL REGULATION OF GLN1 TABLE 1. Yeast strains

Strain

Genotype

Source'

PM38 AM Ta ura3-52 leu2-3, 112 PM71 MATa ura3-52 leu2-3,112 gln3A5::LEU2 PH5 MATa ura3-52 leu2-3,112 ure2A13::LEU2 P. Coschigano a All strains for which no source is listed were constructed in this laboratory for this study.

resulting constructs were screened for orientation of the insert. The plasmid with the insert in the desired orientation contains 552 bp upstream from the GLN1 translational start site and the first 16 amino acids of GLNI fused to lacZ. The junction with lacZ is identical to the larger fusion. The fusions of both constructs were verified by sequence analysis.

Construction of GLNI UAS-CYCI-lacZ fusions. The starting 2,u vector pSLFA178K (10) is a UAS-less CYCJ-lacZ fusion containing three adjacent cloning sites (SmaI, KpnI, and XhoI) which can be used for dropping in putative UAScontaining fragments. The Sal-AvaIl and the HhaI-AvaIl fragments upstream from GLNI (see Fig. 1) were bluntended and cloned into the KpnI (Asp 718) site and the SmaI site, respectively. Construction of GLNJ oligonucleotide-CYCI-lacZ fusions. The GLNI oligomers which were tested for UAS ability were constructed by Stephen Scaringe. The single-stranded oligomers were annealed by heating 20 ,ug of each in 100 pl of annealing buffer (10 mM Tris [pH 7.5], 10 mM MgCl2, 50 mM dithiothreitol) to 60°C for 10 min and then cooling to room temperature over a 45-min period. These oligomers were then phosphorylated by treatment with T4 polynucleotide kinase and cloned into the blunt-ended Asp 718 site of pSLFA178K. Constructs were sequenced to determine number and orientation of inserts. DNA sequencing. DNA sequencing was accomplished by the dideoxy-chain termination method (29) with ao-3S labeling and a modified T7 bacteriophage DNA polymerase, Sequenase (United States Biochemical Corp., Cleveland, Ohio). Subclones for sequencing GLNJ were cloned into Bluescript vectors (Stratagene, La Jolla, Calif.). For the sequencing of GLNJ, double-stranded plasmid DNA templates were prepared by purification over a CsCl gradient (17). Primers used for sequencing GLN1 hybridized to the KS polylinker and were purchased from Stratagene. Both strands of the 1.85-kb SalI-SspI fragment were sequenced by using overlapping subclones. For sequencing constructs used in 13-galactosidase assays, double-stranded Qiagen minipreparations were used as templates. The GLNI-lacZ fusion junctions were sequenced by using a primer which hybridized 17 to 36 nucleotides downstream from the BamHI cloning site; this primer was a gift from L. Guarente. Constructs carrying putative UAS fragments cloned in the CYCI-lacZ vector pSLFA178K were sequenced to determine both the number and orientation of inserts by using a primer which hybridized 82 to 101 nucleotides upstream from the SmaI cloning site. Preparation of DNA probe for Si nuclease mapping. Plasmid pPM69, which contains the 224-bp EcoRI-XbaI GLNI fragment cloned into the Bluescript KS' vector, was digested with EcoRI and XbaI to release the 224-bp fragment encompassing the RNA start site of GLNJ. The purified fragment was treated with alkaline phosphatase and labeled at the 5' end with [-y-32P]ATP and T4 polynucleotide kinase (17).

1829

Si nuclease mapping. The 5' terminus of the GLNJ transcript was mapped by the method of Berk and Sharp (2). The 32P-labeled double-stranded DNA fragment (200 ng) was added to 200 ,g of total cellular RNA in a 20-pl solution of 75% formamide, 40 mM PIPES [piperazine-N,N'-bis(2ethanesulfonic acid); pH 6.4], 0.4 M NaCl, and 1 mM EDTA; heated to 95°C for 4 min; and incubated at 50°C for 4 h. A control experiment was prepared by incubating the same DNA fragment in the absence of RNA and treating it under the same conditions. The hybridization mixture was diluted with 200 ,ul of S1 buffer and incubated at 37°C for 60 min in the presence of 250 U of S1 nuclease. After ethanol precipitation, the pellet was resuspended in 20 pl of 98% formamide plus dye. Portions of 3 or 5 ,ul were run on a 6% polyacrylamide-7 M urea sequencing gel. For a sequencing ladder, Sanger dideoxy-sequencing reactions of pIB16, a Bluescript plasmid containing the SalI-EcoRI fragment of GLN1, were run alongside the S1 reactions. j-Galactosidase assays. Fresh transformants were patched on synthetic complete plates lacking uracil and allowed to grow overnight. These patches were used to inoculate liquid overnight cultures in various media. The overnight cultures were then used to inoculate 10-ml cultures of the same media. The 10-ml cultures were grown to a Klett reading of 90 to 100, and cells were harvested on a Millipore filter. Extracts (final volume, 500 pl) were prepared as described by Mitchell and Ludmerer (21) and were used immediately. ,-Galactosidase assays were done as described by Rose and Botstein (28), and the activity was calculated as described by Miller (18), with the specific activity reported as nanomoles per minute per milligram of total protein. Assays were done with at least 2 volumes of extract, ranging from 10 to 120 ,ul depending on activity. Protein concentrations for all assays were calculated by using the Bradford protein kit (Bio-Rad Laboratories, Richmond, Calif.). Computer methods. Homology searches were accomplished with the FASTA program (25) against a data base which contains NBRF (Georgetown University Medical Center, Washington, D.C.) protein residues and the translation of GenBank (Los Alamos National Laboratories, Los Alamos, N.Mex.) nucleic acid sequences. Nucleotide sequence accession number. The sequence reported here has been deposited in the GenBank data base and has been given accession number M65157. RESULTS Sequencing of GLNI. Both strands of a 1.85-kb SalI-SspI fragment carrying the GLNJ gene were sequenced, revealing one open reading frame which encodes a polypeptide of 370 amino acids with a molecular weight of 41,719 (Fig. 1). This size is consistent with, although slightly smaller than, the reported molecular weight of 43,000 for the purified yeast glutamine synthetase subunit (22). With the exception of two amino acids, the sequences of seven tryptic peptides derived from S. cerevisiae glutamine synthetase (15) are found within the predicted amino acid sequence of the GLNJ gene. A single nucleotide change in two codons of the DNA sequence can lead to the two discrepancies between the peptide sequencing and the amino acid sequence predicted from DNA sequencing. The first of these two discrepancies is found at amino acid residue 165, by our sequencing a glycine encoded by GGT (Fig. 1) but by peptide sequencing an aspartate residue (15) which could be encoded by GAT. Although the change from an aspartate residue to a glycine residue is not a conservative change,

1830

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MINEHART AND MAGASANIK

HhaI

SailI

GTCGACCAGG ATCGCTTCGC ACGTTTGTTT ACAATTGATG ACTGCGCTCC CCTAATAGAT AAGATAAGCT CGCGAAGGCA -581 AvaII GAAAAAAAAA AGTCTTCTAC AGCAGTTGGT CCGCACAGAC GATGCCAGAC GGTGTTTAT CGAAAATTTT TTTCGCATCA -501

TAGTGCCATT TGTGGTCTT ATTATTCCOC AAATATGCGA AAATAGTACA CTATTTTTGG CAGGAGAGTA GGCTGATATG -421 CCGCATTGAT GTCCTGTGTA GCGAAACACA AACAAAAAAA GAAAAAGTAG GATGAAAAA AGAAAAGTAA TATGAAAAAA -341

GAGTGAAAAA TTAATTCATT TGTTAGTGTA AGCGGTCAGG TGTAAGTAGT AGGCTGATA ATGAATTAAA GATGACTCCG -261 ACGCATATTG TTTGCCATGT TTTTATFA GTmTGTAGQT TTCTTTTTTT GTAATATATAIAGGGAGTGAT TCTATATATC -181 AGGTAAGAAC ATCACACAAA GATAACTATA -101

GAATTCTCAG GCTTGGTTGG TTCGTAGGTT

GAATCACATA CATATmTGTG AGAAATTAAC TTCATCAT TTATAGAAGA AGTTCAACCG AAACAAAAAT TAAACATAAT ATAATATAAT R G R I AGAGGTAGAA I T S AATCACATCC Y L K TCTATTTGAA N D G T AATGACGGTA F G L GTTTGGTCTA

ATAATCAAAA I A E TAATTGCCGA I D Q L ATTGACCAAT P V A ACCCGTTGCT P N K CTCCAAACAA E QE Y GAACAAGAAT

M A E A ATGGCTGAAG Y V W ATACGTTTGG P E W TGCCAGAATG Y Y P D TACTACCCAG F N H GTTQAACCAC T L F ACACTCTATT

S I E CAAGCATCGA I D G T ATCGATGGTA N F D GAACTTCGAC P F R ATCCCTTCAG R H E A AGACACGAAG

D M Y TGACATGTAT G K V G V G A GGTGTTGGTG CCGGTAAGGT N A E V S G I TTCTGGTATT AACGCTGAAG A R Y L W M AATTATGGAT GGCCAGATAC N G A K G D W AAGGGTGACT GGAACGGTGC I E K L E Q A CGAACAAGCC ATCGAGAAGT E T A G R H CTGGTAGACA TGAAACCGCT E G Y S V A K TCCZGCGCCA AGGAAGGTTA T V C G M C E CATGTGTGAA ACTGTTTGCG

20 +60 47 140 74 220 100 300 127 380 154 460 180 540 207 620 234 700 260 780 287 860 314 940 340 N I D P CAACATCGAC 1020 367 F E R AATTTGAAAG 1200 370 ACAATAATGC 1212

L D Q ACTGGACCAA K K R TGAAGAAGAG D S D I GACTCTGACA C Y N ATGTTACAAC E I W AAGAATCTG Y P A P TACCCAGCTC R A C CAGAGCTTGT P C T GTCCATGTAC I K I S ATCAAGATCT T K E T N V S M R Q ACTAACGTTT CCACCAAGGA AATGAGACAA Y G S H I K L H A E ACACGCTGAA CACATTAAGT TGTACGGTAG G V A N R G S F S S CCTCTTCTTC TSGTGTOGCC AACAGAGGTA

Y L E I L Q K ATTTTACAAA AATATCTAGA G R T L R S K ACGTTCCAAA GGTAGAACTT P G H N Q A GGTTCTTCTA CCAACCAAGC GCCAGGCCAC L A A N I V V R G D GAGAGGTGAC AACATTGTTG TCTTGGCCGC H K D E F A A A K L CTG=CAAGCT ATTTGCTGCT CATAAGGATG K G G G W P D D V Y GACQTGTTT ACGGATGGCC AAAGGGTGGG A H Y D M I E Y A R TTATGCCAG GACATGATCG AAGCTCACTA F Q V G M P S Q W E TCATGCCATC TCAATGGGAA TTCCAAGTCG V A E E F G F L H R TTTTTGQACA GAGTGGCAGA AGAGTTTGGT

K T Q AAAGACTCAA G N L CTGGrAACTT G S S T

21

Y Y C Q G P CACAAGGTCC TTACTACTGT L E I L Y A G TTGTATGCCG GATTAGAAAT G I D M G D Q CGGTATTGAC ATGGGTGACC G C H K P L F H P CGGTTGTCQC CATTCCATCC AAAGCCATTG S K R K Y I P G G M TATCCAAGAG CCAGGTGGTA TGAAATACAT S M T A D N D M R L T TCCATGACTG CGATAACGAC ATGGATTAA P A S E D R R G X F I P R S I R CGGTTACTTT GAAGACCGTA GACCAGCTTC GCTCAATTAG AATCCCAAGA M T K E N A D A I D Y L V T G I GTGCTATTGA CAATGCTGAC ATGACGAAGG CCATACTTGG TTACAGGTAT * E SS AGAATCTTCA TAAGCATAAT ACAATGGTGC AAAATTTTTT TTAGGCAGGA ACTAATCTAC TAATAAACTA ACAGGAAATA TT FIG. 1. Nucleotide sequence of GLNI and its upstream region. The numbering of the nucleotide sequence is relative to the translational start site. The translated amino acid sequence which encodes a protein of 370 amino acids is shown above the DNA sequence. At position 329, the tyrosine which binds ATP is underlined. Also underlined are the RNA +1 site, 120 bp upstream from the translational start site, and the two putative TATA boxes, 68 and 86 bp upstream from the RNA start site. Restriction sites used in constructs to test upstream activity are indicated at the first nucleotide 3' to the cleavage site.

presumably glutamine synthetase function is unaffected by this change. The second difference, which does represent a conservative change, occurs at position 172. By our sequencing, this residue is an ATG-encoded methionine, but by peptide sequencing this residue was a valine which could be encoded by GTG. The sequence of the N-terminal peptide of glutamine

synthetase as determined by peptide sequencing (15) corresponds to the first seven amino acids after the methionine in the open reading frame of the GLNJ gene, confirming that the start of the open reading frame of the GLNI gene is correct. The peptide sequencing of Kim and Rhee indicated that the protein sequence begins with an acetylated alanine, which corresponds to the alanine after the first methionine in

VOL. 174, 1992

TRANSCRIPTIONAL REGULATION OF GLN1

B

A 1

224

168

1831

2

l

2

3

4

5

6

7

--

--

FIG. 2. S1 nuclease analysis of GLNI transcripts. Total cellular RNA was hybridized to the single-stranded 5'-end-labeled EcoRI-XbaI fragment from -179 to +35 under conditions described in Materials and Methods. The GLNI SaiI-EcoRI fragment was used as a sequencing ladder. (A) Lanes 1 and 2 contain 60 and 100 pLg of RNA isolated from wild-type cells grown on glutamate. (B) Lanes 1 and 2 contain 100 ,ug of RNA isolated from wild-type cells grown on glutamate and on glutamine, respectively. Lanes 3 to 6 contain 100 p,g of RNA isolated from gln3 cells. In lane 3, cells were grown on glutamate without adenine. In lane 4, cells were grown on glutamine and induced with 10 mM 3-AT for 0.6 generations. In lane 5, cells were grown on glutamate plus adenine, and in lane 6, cells were grown with glutamine. Lane 7 shows the probe carried through the Si procedure in the absence of RNA.

our sequence. Kim and Rhee also sequenced the tryptic peptide containing the ATP-binding site, but they could not be certain of the amino acid which covalently binds ATP, because it was modified in their purification procedure. They proposed, and our sequence confirms, that this amino acid is a tyrosine residue, found at position 329 (Fig. 1). Si nuclease mapping of the GLN1 transcript. Si nuclease mapping indicated one discrete size family of transcripts, with 5' termini centered at an adenine residue 120 bp upstream from the translational start site (Fig. 1 and 2). The start site used in wild-type cells was used in gln3 cells. This start site did not change when cells were grown with glutamate rather than glutamine as the source of nitrogen. Neither adenine limitation nor histidine starvation altered the GLNJ start site. The level of GLNI transcript did increase when cells were grown under inducing conditions, confirming earlier results that GLNI is controlled at the level of transcription (1) (Fig. 2). Two potential TATA boxes, both with the sequence TATATA, can be found, one 68 bp upstream from the transcriptional start site and the other 86 bp upstream from the transcriptional start site (Fig. 1). Upstream analysis of the GLNI gene using GLNI-lacZ fusions. In order to define cis-acting sites involved in the regulation of the GLNI gene, the region encoding the first 16 amino acids of GLN1 and 659 bp upstream of the GLNJ translational start site (5' end at the Sall site in Fig. 1) were fused to the lacZ gene in the 2,u vector pSLFA178K (13). Earlier analysis using a GLNI-lacZ fusion in which lacZ was fused much further downstream in the GLNI gene had shown that this upstream region contained all the informa-

tion required for regulation of GLNI in response both to a shift from glutamine to glutamate as the nitrogen source and to purine limitation (1). Our results with the early GLN1lacZ fusion confirm these results. The increase in response to the nitrogen source can be seen by comparing 0-galactosidase levels in wild-type cells grown on glutamate with ,-gaIactosidase levels in wild-type cells grown on glutamine. P-Galactosidase levels in glutamate-grown cells are almost 100-fold higher than P-galactosidase levels in glutamine-grown cells. Our results further show that this 100-fold increase with glutamate depends on the presence of the GLN3 gene (Table 2). Although the strain background is Ade+, adenine is included in the minimal growth media to eliminate activation by purine limitation. Earlier work has shown that although levels of glutamine synthetase in wild-type cells are unaffected by the presence of adenine in the growth medium, levels of glutamine synthetase in gln3 cells are reduced when adenine is added to glutamate-grown cells (20). Genetic analysis has shown that, in addition to GLN3, another gene, URE2, is involved in the glutamine-glutamate regulation of glutamine synthetase. While mutations in GLN3 lead to low levels of glutamine synthetase, mutations in URE2 lead to high levels in both glutamate- and glutamine-grown cells (8, 11, 31). Since a gln3 ure2 double mutant has the phenotype of a gln3 mutant, the URE2 product is thought to inactivate the GLN3 product (5, 6, 20). The effect of the ure2 mutation on ,B-galactosidase levels in cells carrying the GLNJ-lacZ fusion parallels the effect of the ure2 mutation on glutamine synthetase. That is, the

1832

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MINEHART AND MAGASANIK

Plasmid

SalIc (-659 to +1) AvaIIC (-552 to +1)

SalIlAvaIIe (-659 to -550, forward orientation) SalIlAvaIIe (-659 to -550, reverse orientation)

TABLE 2. P-Galactosidase activity of GLNI-lacZ translational fusions Sp act (nmol/min/mg of protein) in the following growth medium': Straina

WT gln3 ure2 WT gln3 ure2 WT gln3 ure2 WT gln3 ure2

GLN

GLN + 3-AT

71 + 9 92 8 5,321 ± 1,017 147 ± 15 148 23 1,049 100 10 ± 1 ND 540 ± 63 10 ± 1 ND 5,035 ± 343

227 ± 42 112 ± 16 NDd 146 ± 20 198 ± 23 ND ND ND ND ND ND ND

GLT

6,685 81 7,341 241 84 758 907 45 858 1,134 46 6,596

± 815

20 + 1,023 ± 48

+

± ± ±

±

19 70 92 2 106 146 3 113

GLT - Ade

6,479 ± 832 ND ND 534 ± ND ND 49 + ND ND 37 ± ND

631 24

111 4 10

a PM38 was used as the wild type (WT) strain, PM71 was used as the gln3 strain, and PH5 was used as the ure2 strain. b Mid-exponential phase cultures growing in the indicated minimal medium with either glutamine (GLN) or glutamate (GLT) as the nitrogen source were harvested, and l-galactosidase specific activity was determined. Where indicated, the histidine analog 3-AT was added to the glutamine medium at a final concentration of 10 mM for a duration of 0.6 generations. Adenine (Ade) was added to a final concentration of 0.15 mM to both the glutamate and glutamine media except where indicated (rightmost column). Values indicated are the averages of at least two determinations for each of three transformants. c Cells carried one of the two GLNI-lacZ translational fusions with 5' end points as indicated. In addition to the indicated upstream region, these fusions contain the first 16 amino acids of GLNI. As a control, the CYCI-lacZ vector pSLFA178K was also tested. It gave less than 5 U of activity in each of the three cell backgrounds, under all conditions. d ND, not determined. e The indicated restriction fragment from GLNI upstream DNA was cloned into pSLFA178K, the CYCI-lacZ vector missing upstream activation sequences. Orientation describes the direction of the GLNI insert with respect to the CYCI start site. In the forward orientation, the GLNI fragment lies in the same direction as in the wild-type GLN1 upstream sequence, while in the reverse orientation, the GLNJ fragment lies in the opposite orientation than in the GLNI upstream sequence.

0-galactosidase level in glutamine-grown ure2 cells was greatly increased and was approximately equivalent to the ,B-galactosidase level in glutamate-grown wild-type cells. 1-Galactosidase levels in glutamate-grown cells were only slightly increased by the ure2 mutation, so that in a ure2 background, levels in glutamine-grown cells were nearly the same as in glutamate-grown cells (Table 2). The increase in response to purine limitation can be seen by comparing ,B-galactosidase levels of the gln3 strain grown on glutamate in the absence of adenine with ,-galactosidase levels of the gln3 strain grown on glutamate in the presence of adenine, since gln3 strains grown on glutamate without adenine are starved for purines (20). Activation by purine starvation is significantly weaker than activation by the replacement of glutamine with glutamate as the nitrogen source. Our results with the GLNI-lacZ fusion showed that ,-galactosidase activity was approximately 10-fold higher in gln3 cells grown on glutamate in the absence of adenine than in those grown in the presence of adenine (Table 2). This upstream region also was reported to have the necessary information for the response to histidine starvation via GCN4 (1). With the new fusion, 3-aminotriazole (3-AT)-induced histidine starvation did lead to a discernible increase in ,-galactosidase levels in a GLN3 strain; however, no increase could be seen in a gln3 strain (Table 2). Previous results had shown that in a GLN3 background, most of the effect on GLNI caused by histidine starvation was due to indirect activation by GLN3 as a result of the depletion of glutamine pools caused by increased histidine biosynthesis (24). This is reflected in the ability to see a 3-AT-induced increase in ,B-galactosidase only in the GLN3+ strain. Our inability to see an effect on the GLNJ-lacZ fusion in a gln3 strain does not prove, however, that the GCN4binding site is further upstream in the chromosomal DNA, since the GCN4 effect on chromosomally encoded glutamine synthetase is quite small in gln3 strains (24) and the 3-galactosidase assay using high-copy plasmids may not be sensi-

tive enough to reveal this difference. Further indication that the sequences necessary for the GCN4-mediated response are contained within this fusion comes from the fact that the consensus sequence for the GCN4-binding site (TGACTC) is found at -268 relative to the translational start site (Fig. 1). We next constructed a GLNI-lacZ fusion which contained only 552 bp upstream of the translational site (5' end at the AvaIl site in Fig. 1). This fusion was still regulated in response to adenine limitation, but only a small increase in f-galactosidase was seen when glutamine was replaced by glutamate as the sole nitrogen source (Table 2). These results indicate that the sequences necessary for purine control are contained within 552 bp upstream of the translational start site and that some of the sequences necessary for GLN3mediated control are found between 552 and 659 bp upstream of the translational start site. The small increase seen when glutamate replaces glutamine as the nitrogen source for cells carrying the GLNIlacZ fusion deleted to theAvaII site indicates that GLN3 still has a slight effect on the activity of this fusion. This idea is further substantiated by the effect of the ure2 mutation on this fusion, which increased 3-galactosidase to a level 7- to 10-fold higher than the level in the gln3 strain (Table 2). The ability of the Sall-Avall fragment (-659 to -550) to confer ,B-galactosidase expression regulated by the nitrogen source was examined by the use of a plasmid in which this DNA fragment had been fused in both orientations to CYCl-lacZ. This fragment was still capable of conferring a regulated ,B-galactosidase in response to the nitrogen source (Table 2). Both the wild-type glutamine basal level and the induced glutamate level were decreased, but the regulation was still strong since the level of 3-galactosidase in wild-type cells grown with glutamate as the nitrogen source was approximately 100-fold higher than the level in wild-type cells grown with glutamine as the nitrogen source. This elevated level of ,-galactosidase in glutamate-grown cells was dependent on the presence of the GLN3 gene product.

TRANSCRIPTIONAL REGULATION OF GLN1

VOL. 174, 1992

1833

TABLE 3. ,B-Galactosidase levels in cells carrying CYCI-lacZ fusions to sequences within the GLNI nitrogen UAS'

13-Galactosidase DNA

of copies and orientation No.

GLT

HhaI-AvaII (-614 to -550)

Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide pSLFA178K (CYCI-lacZ vector)

1 1 1

2 2

3 J- 4 7 4- 4 0

sp act

(nmol/min/mg) ure2

Wild type

26 19 11 105 107 731 642 3

± ± ± ± t

± ± ±

5 2 5 14 21 103 15 2

GLN

GLT

GLN

4 ± 1 3 +1 2 ± 1 2 t 1 2 ± 1 2 ± 1 9 ± 2 4t 2

101 ± 5 187 ± 9

150 ± 33 257 ± 31 ND ND ND 1,055 t 97 1,071 ± 95 5 ± 3

NDb ND ND 830 ± 99 655 t 74 2t 2

a Arrows indicate the direction and the position of the GLNI oligonucleotide sequence (GCGCTCCCCTAATAGATAAGATAAGCTCGCGA; 32 bp, -617 to -586). Where present, a number over an arrow indicates the number of sequential repeats. Mid-exponential phase cultures growing in the indicated minimal medium (minimal medium with glutamate [GLT] or glutamine [GLN]) were harvested, and 13-galactosidase specific activities were determined. Adenine was added to a final concentration of 0.15 mM to both the glutamate and the glutamine media. Values indicated are the averages of at least two determinations for each of three transformants. b ND, not determined.

Thus, the sequences of the 109-bp Sall-AvaII restriction fragment are sufficient for GLN3-mediated transcriptional control of GLN1 in response to the replacement of glutamine by glutamate as the sole nitrogen source. Furthermore, the Sall-AvaIl fragment satisfies the definition of a UAS since it works in both orientations (12). As the sites involved in purine control lie downstream from the AvaII site, the GLN1 nitrogen UAS is separate from the purine control site. The 3-galactosidase activity conferred by the Sal-Avall fragment in wild-type cells grown on glutamate was approximately sixfold lower than the activity conferred by the entire upstream region, indicating that some sequences downstream of the AvaIl site are capable of increasing the GLN3-mediated activity. Presumably, these are the same sequences responsible for the small GLN3-dependent activation seen in the GLNJ-lacZ fusion deleted to the AvaII site. Although the Sall-AvaIl fragment is missing these sequences, the activity conferred by this fragment could be pushed to the higher level by the presence of the ure2 mutation when the fragment had been inserted in the reverse orientation. Thus, this higher level of ,B-galactosidase can be achieved either by the presence of additional sites or by the ure2 mutation. We do not know why this effect is not seen when the fragment is inserted in the forward orientation. Further delineation of the GLN1 nitrogen UAS. When cloned into the CYCJ-lacZ vector, a HhaI-AvaII restriction fragment (-614 to -550) which deletes 45 bp from the 5' Sall site gave low ,-galactosidase activity in the wild-type cells grown on either glutamate or glutamine, with the level in glutamate-grown cells being slightly higher than the level in glutamine-grown cells (Table 3). Since the 3-galactosidase level decreased so sharply from the level conferred by the Sal-AvaIl fragment, the 5' end point of the GLNI nitrogen UAS must be within the 44 bp between the SalI site and the HhaI site. However, the HhaI-AvaII restriction fragment contains the sequence GATAA, which has been shown to be important in the regulation of other GLN3-regulated genes (2a, 4, 4a, 27, 33). Therefore, we tested a 32-bp oligonucleotide which consists of the sequence -617 to -586 and contains the repeated GATAA motif for UAS activity (Table 3). The results in Table 3 show that one copy of the oligonucleotide in either orientation was not sufficient for significant activation of the CYCl-lacZ fusion, but two oligonucleotide copies did provide regulated UAS activity. Two copies of this

oligonucleotide did not increase the 3-galactosidase level to the level supported by the Sall-AvaIl restriction fragment. However, p-galactosidase activity conferred by three copies of the oligonucleotide approximated the level provided by the Sall-AvaIl fragment. Increasing the copy number of the oligonucleotide to seven did not lead to a further increase in ,-galactosidase activity. The UAS activity of these oligonucleotides was regulated by the nitrogen source (Table 3). The UAS activity of the sevenfold repeat of the shorter oligonucleotide was tested in a gin3 background, and the increase with glutamate was shown to be dependent on the presence of the GLN3 gene (data not shown). Thus, although the oligonucleotide as a single copy does not contain all the information necessary for the full nitrogen response, when present in multiple copies, it is capable of eliciting a clear response to the nitrogen source. Just as increasing the copy number of the oligonucleotide led to higher levels of ,B-galactosidase activity, the presence of the ure2 mutation increased P-galactosidase levels conferred by both the single oligonucleotide and the HhaI-AvaII fragment in both glutamate-grown and glutamine-grown cells (Table 3). That the P-galactosidase activity of these constructs could be pushed higher either by the ure2 mutation or by increasing the copy number indicates that the GLN3binding site is contained within these sequences. As these sequences by themselves were not sufficient for the complete activation in response to the replacement of glutamine by glutamate as the source of nitrogen, the GLNI promoter must also contain some auxiliary sequences involved in the response. Since the HhaI-AvaII fragment gave ,B-galactosidase levels similar to those of the oligonucleotide, the additional activation seen with the Sal-AvaII fragment must be due to sequences between the SalI site and the HhaI site. The limit in ,B-galactosidase activity seen with ure2 cells carrying the forward orientation of the SalI-AvaIl fragment was also seen with the constructs carrying multiple copies of the oligonucleotide (Table 3). That is, in glutamate-grown strains carrying either the construct with three copies of the oligonucleotide or the construct with seven copies of the oligonucleotide, an increase in P-galactosidase activity caused by the ure2 mutation was not seen. This indicates that the higher levels seen with the reverse orientation of the constructs must be attributed to sequences outside of the major GLN3-binding site.

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DISCUSSION We have sequenced GLN1, the gene encoding glutamine synthetase. The predicted amino acid sequence of GLNI is highly homologous to those of other eukaryotic glutamine synthetases, having greater than 50% identity in an overlap region of 340 amino acids. It is less homologous to the glutamine synthetase of E. coli and Salmonella typhimunum, having only 30% identity in an overlap of 170 amino acids (25). By sequence analysis, the predicted molecular weight is approximately 42,000, which is consistent with the reported molecular weight of 43,000 for the purified glutamine synthetase subunit. This monomer weight still indicates that the active multimer, which has a molecular weight of 470,000, must consist of 10 to 12 subunits. This seems to be unique to S. cerevisiae, as all other eukaryotic glutamine synthetases studied have been octamers. Since prokaryotic glutamine synthetases are dodecamers, the overall structure of S. cerevisiae glutamine synthetase may be closer to those of prokaryotic glutamine synthetases than are other eukaryotic glutamine synthetases. A previous comparison of glutamine synthetases has shown that five amino acid residues which have been shown to act as ligands to the Mn 2+ ion in S. typhimunium are conserved among both prokaryotes and eukaryotes (26). These amino acids-the glutamate at position 133, the glutamate at position 193, the phenylalanine at position 201, the histidine at position 250, and the glutamate at position 330-are also conserved in the S. cerevisiae glutamine synthetase. The tyrosine at position 328, which binds ATP, is conserved among eukaryotic glutamine synthetases but not prokaryotic glutamine synthetases. It is, however, at the beginning of the last of five domains which, as shown by the alignment of Pesole et al. (26), is conserved among prokaryotes and eukaryotes and contains an ATP-binding site for both. Glutamine synthetase is regulated not only at the level of transcription but also by glutamine-dependent inactivation of the enzyme (23). Although this inactivation has the same effect as the inactivation caused by adenylylation in enteric bacteria, no evidence for the adenylylation system has been found in yeasts. Preliminary evidence has indicated that the inactivation instead may involve modification of sulfhydryl groups (15). Our sequence analysis indicates that seven cysteine residues are present in the GLNI protein, but as yet the cysteine residues involved in this inactivation have not been identified. Si nuclease mapping has indicated that the RNA start site is 120 bp upstream from the translational start site and that this start site does not change irrespective of the condition used to activate the transcription of GLN1. Sequence analysis showed that two potential TATA boxes, separated by 18 bp, exist. One is 68 bp upstream from the transcriptional start site and the other is 86 bp upstream from the transcriptional start site. Three separate systems control the transcription of the GLNJ gene. The weakest of these regulations is determined by the general amino acid control system. Upstream analysis has not delineated a specific region involved in general amino acid control because this control is apparently too weak to be seen by using lacZ fusions, but sequence analysis does show a GCN4-binding site at -268 to -264 (Fig. 1). Regulation which responds to starvation for purines is slightly stronger than the regulation by GCN4, and the sites involved in purine control have been shown to be located downstream of the sites involved in nitrogen source regula-

J. BACTERIOL.

tion. The strongest regulation exerted on the GLNI gene is the URE2/GLN3 system, which activates the expression of GLNI in response to glutamine starvation. The major GLNJ upstream sequences determining this response lie between 550 and 659 bp from the translational start site, upstream from the sequences determining the response to purine starvation. We have shown that the SalI-AvaI fragment containing these activating sequences (-659 to -550) is indeed bound by the GLN3 protein (20). This region can activate GLNI transcription in the reverse orientation as well as in the forward orientation and thus satisfies the definition for a UAS. Within the 109-bp region that contains the nitrogen UAS is a 32-bp sequence that does not contain all the information necessary for the nitrogen control response, as it does not allow strong activation when present as a single copy but does appear to bind a transcription factor, since it can activate the CYCl-lacZ fusion either when present in multiple copies or in the absence of the URE2 gene product. This activation is regulated by the nitrogen source and is dependent on GLN3. Within this region, between -603 and -593, is the sequence 5'-GATAAGATAAG-3'. A homologous sequence, 5'-GATTAGATTAG-3', is also found in the region upstream of GDH2 and has been shown to be responsible for the GLN3-dependent response to nitrogen regulation of that gene (19). This GATAA motif had been identified earlier in the upstream activating sequences of two other genes which respond to GLN3, namely, DAL7 and DALS (27, 33). As this sequence is also a subset of the consensus binding site for the animal GATA-1 factor that has zinc fingers homologous to the zinc finger of GLN3 (9), it seemed likely that this sequence was the binding site for GLN3. In fact, GLN3 was shown to bind this 32-bp oligonucleotide in the sevenfold repeat shown in Table 3 (20). The activity of the HhaI-AvaII fragment is similar to the activity of the single oligonucleotide. Not only are 0-galactosidase levels in wild-type cells grown on either glutamate or glutamine similar for the two constructs, but the ure2 mutation also boosts the activity similarly for each construct. For both constructs, the level of 0-galactosidase in wild-type cells grown on glutamate is significantly lower than the level for the Sall-AvaII fragment. Since the sequences downstream from the oligonucleotide which are found in the Sail-AvaIH fragment are also found in the HhaI-AvaII fragment, the higher activation levels of the Sall-AvaII fragment must be due to sequences upstream from the HhaI site. Presumably, the additional activation seen with the SallAvaIl fragment is caused by the binding of a second factor. This second factor does not have to be directly involved with GLN3 or part of the GLN3/URE2 regulatory pathway, as recent work has shown that eukaryotic activators do not have to interact with each other in order to stimulate transcription cooperatively (3, 16). It has been shown that the response of another gene, GDH2, to GLN3 depends both on sequences homologous to GATAA and on an auxiliary element. This auxiliary element contains a sequence of nucleotides, 5'-TTGGT11T-3', homologous to the GLNI upstream sequence 5'-1TTGT-11T-3' located upstream of the HhaI site between -517 and -525. An important difference between the regulation of GDH2 expression and GLNJ expression is the response to the deletion of the URE2 gene. In glutamate-grown cells, deleting URE2 leads to an approximately fivefold increase in the GDH2-encoded NAD-linked glutamate dehydrogenase activity as well as in the 3-galactosidase activity conferred by both a complete GDH2-1acZ fusion and a GDH2 nitrogen

VOL. 174, 1992

UAS-lacZ fusion (5, 19). Deleting URE2 in glutamate-grown cells also leads to an approximately fivefold increase in 1-ga-

lactosidase activity conferred by the GLNI nitrogen UAS-lacZ fusion. However, the level of GLNI-encoded glutamine synthetase (5) and of 1-galactosidase conferred by a complete GLNJ-lacZ fusion is only slightly increased by the absence of URE2 in glutamate-grown cells. Since expression conferred by the 109-bp GLNJ nitrogen UAS parallels the expression conferred by both the GDH2 UAS and the complete GDH2 gene, but not the expression conferred by the complete upstream sequences of the GLNI gene, the difference between GDH2 and GLNI in the response to the ure2 deletion must lie in the GLNJ sequences downstream of its nitrogen UAS. This region, downstream of the AvaII site, was shown to allow weak GLN3-dependent activation, indicating that other GLN3-binding sites are probably present. In fact, the GATAA motif is found three times in the region between the AvaII site and the transcriptional start site. In the forward orientation, the sequence GATAT is found between -426 and -422, while the sequence GATAA is found between -284 and -280. Looking at the opposite strand, from right to left, the sequence GATAA falls between -520 and -524. These extra sequences, which are not found outside of the GDH2 UAS, may explain the ability of the GLNI gene to reach its maximal activity in wild-type glutamate-grown cells, whereas the GDH2 gene is unable to reach its maximal activity unless it receives the additional stimulation conferred by the ure2 deletion. ACKNOWLEDGMENTS We thank the members of the Magasanik laboratory, especially Peter Coschigano, for helpful suggestions and discussions. We also thank Stephen Miller for assistance in some plasmid constructions, Jae-Min Rhee for assistance in sequencing, and Itai Barel for some initial deletion analysis. This work was supported by Public Health Service research grant GM-07446 to B.M. P.L.M. was supported by a National Institutes of Health training grant and an Amoco fellowship. REFERENCES 1. Benjamin, P. M., J.-L. Wu, A. P. Mitchell, and B. Magasanik. 1989. Three regulatory systems control expression of glutamine synthetase in Saccharomyces cerevisiae at the level of transcription. Mol. Gen. Genet. 217:370-377. 2. Berk, A. J., and P. A. Sharp. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonucleasedigested hybrids. Cell 12:721-732. 2a.Bysani, N., J. R. Daugherty, and T. G. Cooper. 1991. Saturation mutagenesis of the UASNTR (GATAA) responsible for nitrogen catabolite repression-sensitive transcriptional activation of the allantoin pathway genes in Saccharomyces cerevisiae. J. Bacteriol. 173:4977-4982. 3. Carey, M., Y.-S. Lin, M. R. Green, and M. Ptashne. 1990. A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. Nature (London) 345:361-364. 4. Cooper, T. G., D. Ferguson, R. Rai, and N. Bysani. 1990. The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae. J. Bacteriol. 172:1014-1018. 4a.Cooper, T. G., R. Rai, and H. S. Yoo. 1989. Requirement of upstream activation sequences for nitrogen catabolite repression of the allantoin system genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 9:5440-5444. 5. Coschigano, P. W., and B. MagasaniL 1991. The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to nitrogen source and has homology to glutathione S-transferases. Mol. Cell. Biol. 11:822-832. 6. Courchesne, W. E., and B. Magasanik. 1988. Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes. J. Bacteriol. 170:708-713.

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