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Volume 272, Number 20, Issue of May 16, 1997 pp. 13343-13354
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7*

(Received for publication, December 3, 1996, and in revised form, February 25, 1997)

Ronda J. Rolfes Dagger §, Fan Zhang and Alan G. Hinnebusch

From the Dagger  Department of Biology, Georgetown University, Washington, D. C. 20057 and the  Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves down-regulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.

INTRODUCTION

The de novo synthesis of purine nucleotides requires 10 enzymatic steps to form the first purine nucleotide, inosine monophosphate. IMP is converted to either AMP or GMP in two steps. The products of at least 13 genes are required for this synthesis, since mutations in any one of these lead to an adenine requirement (1). Most of these genes encode the biosynthetic enzymes that participate in specific steps of the pathway (ADE1, ADE2, ADE4, ADE5,7, ADE6, ADE8, ADE12, ADE13), others encode enzymes required to produce additional substrates necessary to complete the pathway (ADE3, ASP5), and the function of one gene product is unknown (ADE9). Strains with mutations in two additional genes, BAS1 and BAS2, have a partial adenine requirement (2). In these latter mutants, expression of the biosynthetic enzymes is low, indicating a positive regulatory role for these gene products (3, 4).

Expression of the adenine biosynthetic genes is repressed when cells are grown in the presence of adenine (3-6). This adenine-mediated repression occurs at the transcriptional level (7). ADE gene transcription is unregulated in bas1 or bas2 mutant strains (3). The expression of BAS1 and BAS2 is not regulated by adenine levels (8, 9), however, indicating that adenine repression occurs by down-regulating the activator functions of the BAS1 or BAS2 proteins.

BAS1 and BAS2 were identified as transcriptional activator proteins required for the basal expression of the HIS4 gene (2). BAS1 binds to DNA using an amino-terminal myb motif (8). This tryptophan-rich motif is repeated three times in the BAS1 protein (8, 10). BAS1 binds to two sites in the HIS4 promoter (8) and to two sites in each of the promoters for the ADE2 and ADE5,7 genes (3). The consensus sequence derived from these six BAS1 binding sites is TGACTC. Interestingly, this hexanucleotide sequence forms the core of the binding site for GCN4 protein as well, although flanking nucleotides differently affect the binding affinity of these two proteins (10, 11). Whereas GCN4 has a preference for the sequence RRTGACTCATTT (R represents A or G; Ref. 11), none of the known BAS1 sites or any predicted sites in other ADE gene promoters have either an A nucleotide at the 3' base following the conserved hexanucleotide core or any other sequence conservation.

BAS2 binds to DNA via an amino-terminal homeodomain that is closely related to the engrailed protein of Drosophila (8). The BAS2 binding site at HIS4 has been mapped to an A + T-rich repeat, TTAA (8). The results of electrophoretic mobility shift assays suggested that BAS2 binds to the ADE2 and ADE5,7 promoters; however, no specific binding sites were identified (3). In addition to participating with BAS1 in transcriptional activation of HIS4 and the ADE genes, BAS2 (also known as PHO2 and GRF10) stimulates transcription of PHO5 (12) and HO (13). High level transcription of HO requires BAS2 in conjunction with SWI5 (13, 14). Binding of BAS2 and SW15 to the HO promoter is cooperative in vitro (13, 15). BAS2 also interacts with PHO4 to induce PHO5 transcription under phosphate starvation conditions (16). Binding of PHO4 is BAS2-dependent and restricted to derepressing conditions as shown by in vivo footprinting experiments (17). There is evidence that BAS2 and PHO4 physically interact at the promoter and that this interaction is regulated by phosphate (18).

Because expression of the genes that are activated by BAS1 and BAS2 is lower when cells are grown in adenine excess, adenine antagonizes the activation function of one or both of these proteins. Excess adenine could inhibit their ability to interact with one another, to bind to DNA, or to interact with components of the transcriptional machinery. Down-regulation of BAS protein function could also occur by covalent modification or through binding of a negative regulator, analogous to binding of the repressor GAL80 to the activator GAL4 (12). Alternatively, a DNA binding repressor might interact with negative control sites in the promoter, as described for the MIG1-TUP1-SSN6 complex that mediates the carbon catabolite repression of GAL genes (12).

To define more clearly the cis- and trans-acting regulatory elements required for adenine regulation, we performed an extensive analysis of the ADE5,7 promoter. First, we identified the minimal sequences from ADE5,7 sufficient to confer adenine-regulated activation (UASADE5,71 function) to a heterologous CYC1-lacZ reporter lacking its native UASCYC. We then subjected the minimal UASADE5,7 to extensive mutagenesis by making successive 3-nucleotide substitutions across this element to identify precisely the critical nucleotides contained therein. Our results indicate that of the two BAS1 binding sites at UASADE5,7, the gene-proximal copy is the more critical, being both necessary and sufficient for adenine-regulated promoter activity. Two additional regions were essential for UASADE5,7 function. One region, located 5' to the critical BAS1 site, is a binding site for ABF1. The other, located 3' to the critical BAS1 site, is an extended A + T-rich element that appears to be a Bas2 binding site. We found that none of the nucleotide substitutions in the UASADE5,7 led to constitutively derepressed expression, the phenotype expected from the loss of a repressor binding site. Thus, our results suggest that the repression of UASADE5,7 function in adenine-replete cells involves a modification of one of the three transcriptional activator proteins, BAS1, BAS2, or ABF1, rather than binding of a repressor.

EXPERIMENTAL PROCEDURES

Strains and Media

Saccharomyces cerevisiae strains AY854 (alpha  GCN4 BAS1 BAS2 ura3-52), AY856 (a GCN4 bas1-2 BAS2 ura3-52), AY858 (alpha  GCN4 BAS1 bas2-2 ura3-52), AY860 (alpha  GCN4 bas1-2 bas2-2 ura3-52), AY957 (a gcn4 BAS1 BAS2 ura3-52), and AY862 (a gcn4 bas1-2 bas2-2 ura3-52) were provided by Kim Arndt (Cold Spring Harbor Laboratories). ElectroMax Escherichia coli strain DH10B (Life Technologies, Inc.) was used for transformation (19).

E. coli cells were grown in LB medium supplemented with 100 µg/ml ampicillin. S. cerevisiae cells were grown in synthetic dextrose (SD) medium (20) supplemented with 0.5 mM arginine and 0.3 mM histidine. Adenine was added to 0.15 mM in the cultures grown under repressing conditions.

Oligonucleotides and Plasmids

Table I lists the oligonucleotides that were used as polymerase chain reaction (PCR) and sequencing primers for cloning and as probes for DNA binding assays as described below. Deletion of an XhoI fragment that contained the UASCYC1 from plasmid pLG699Z (21) generated pLG699ZDelta XhoI. Plasmid pCM81, a derivative of pLG699ZDelta XhoI, contains an oligonucleotide that replaces the unique XhoI site with adjacent BglII and XhoI sites (22). Fragments of the ADE5,7 promoter were generated by the PCR using plasmid pYEADE5,7(5.2R) (23) and the primers listed in Table I, as described in the relevant figure and table legends. PCR reactions were performed using Taq polymerase (Perkin-Elmer) under the following conditions: 30 cycles of denaturing at 95 °C for 2 min, annealing at 55 °C for 1 min, and chain extension at 72 °C for 30 s. PCR fragments were digested with either XhoI or XhoI and BglII, separated from primers by electrophoresis, purified, and inserted into pLG699ZDelta XhoI or pCM81. The ligated products were transformed into competent E. coli by electroporation (19). Bacterial transformants were screened for insertions by colony hybridization (24), by restriction analysis (24), or by whole cell PCR (25). The nucleotide sequence of the vector-insert junctions was verified in all constructs by sequence analysis using primer RO-26, which corresponds to positions -160 to -142 (relative to the ATG codon) of the CYC1 gene. Plasmids were transformed into yeast cells using lithium acetate (26) and plated on medium lacking uracil.

Table I. Oligonucleotides

Sequences of the oligonucleotides are listed from their 5' end. Comments indicate what the oligonucleotide was used for. Underlined positions indicate restriction enzyme sites used for cloning, as indicated in the text. seq., sequencing primer; mut., mutant. Sequences of the oligonucleotides are listed from their 5' end. Comments indicate what the oligonucleotide was used for. Underlined positions indicate restriction enzyme sites used for cloning, as indicated in the text. seq., sequencing primer; mut., mutant.
Name Comments Sequence
RO-26 CYC1 seq. 5'-GCCATATGATCATGTGTCG-3'
RO-27 End -271 5'-CGTCCTCGAGGTGGCAGTAAGCAGC-3'
RO-28 End -133 5'-CGTCCTCGAGGTTCAAGCCCATCGC-3'
RO-29 End -233 5'-CGTCCTCGAGCATTTTTTTTTTCAGTTGAC-3'
RO-30 End -160 5'-CGTCCTCGAGGCTGTTATTACCAGGACACG-3'
RO-35 Distal site 5'-CGTCCTCGAGCATTTTTTTTTTCAGTTGAATTCGCC-3'
RO-36 Proximal site 5'-CGTCCTCGAGGCTGTTATTACCAGGACACGAATTCAG-3'
RO-39 End -211 5'-CGTCCTCGAGGCCCCGTCGGTAGTG-3'
RO-40 End -183 5'-CGTCCTCGAGGTGGGCACTTGTCAC-3'
RO-41 End -145 5'-CGTCCTCGAGCGCATATTCATTATTGC-3'
RO-43 End -195 5'-CGTCCTCGAGCAAGTGCCGACTGACTC-3'
RO-53 End -211 5'-CGTCAGATCTGCCCCGTCGGTAGTGAC-3'
RO-54 Cluster 1 5'-CGTCAGATCTNNNCCGTCGGTAGTGAC-3'
RO-55 Cluster 2 5'-CGTCAGATCTGCCNNNTCGGTAGTGAC-3'
RO-56 Cluster 3 5'-CGTCAGATCTGCCCCGNNNGTAGTGAC-3'
RO-57 Cluster 4 5'-CGTCAGATCTGCCCCGTCGNNNGTGACAAG-3'
RO-58 Cluster 5 5'-CGTCAGATCTGCCCCGTCGGTANNNACAAGTGCC-3'
RO-59 Cluster 6 5'-CGTCAGATCTGCCCCGTCGGTAGTGNNNAGTGCCGA-3'
RO-60 Cluster 7 5'-CGTCAGATCTGCCCCGTCGGTAGTGACANNNGCCGACTG-3'
RO-61 Cluster 8 5'-CGTCAGATCTGCCCCGTCGGTAGTGACAAGTNNNGACTGACT-3'
RO-62 Cluster 9 5'-CGTCAGATCTGCCCCGTCGGTAGTGACAAGTGCCNNNTGACTCGT-3'
RO-63 Cluster 10 5'-CGTCAGATCTGCCCCGTCGGTAGTGACAAGTGCCGACNNNCTCGTGTC-3'
RO-64 Cluster 11 5'-CGTCAGATCTGCCCCGTCGGTAGTGACAAGTGCCGACTGANNNGTGTCCTG-3'
RO-65 Cluster 12 5'-CGTCCTCGAGCGCATATTCATTATTGCTGTTATTACCAGGANNNGAGTCAGT-3'
RO-66 Cluster 13 5'-CGTCCTCGAGCGCATATTCATTATTGCTGTTATTACCANNNCACGAGTCAG-3'
RO-67 Cluster 14 5'-CGTCCTCGAGCGCATATTCATTATTGCTGTTATTANNNGGACACGA-3'
RO-68 Cluster 15 5'-CGTCCTCGAGCGCATATTCATTATTGCTGTTANNNCCAGGACA-3'
RO-69 Cluster 16 5'-CGTCCTCGAGCGCATATTCATTATTGCTGNNNTTACCAGG-3'
RO-70 Cluster 17 5'-CGTCCTCGAGCGCATATTCATTATTGNNNTTATTACCAGG-3'
RO-71 Cluster 18 5'-CGTCCTCGAGCGCATATTCATTANNNCTGTTATTACCAGG-3'
RO-72 Cluster 19 5'-CGTCCTCGAGCGCATATTCANNNTTGCTGTTATTACC-3'
RO-73 Cluster 20 5'-CGTCCTCGAGCGCATATNNNTTATTGCTG-3'
RO-74 Cluster 21 5'-CGTCCTCGAGCGCANNNTCATTATTGCTG-3'
RO-75 Cluster 22 5'-CGTCCTCGAGNNNNTATTCATTATTGCTG-3'
RO-97 Linker 5'-GATCGTCCATATGCTCG-3'
RO-98 Linker 5'-GATCCGGAGCATATGGAC-3'
RO-103 BAS2 5'-GATCGAGATGATGGAAT-3'
RO-104 BAS2 5'-AATTCTTCCATCATCTC-3'
RO-107 BAS1 5'-CATTTTATCGCATATGTCGAATATAAGTACC-3'
RO-108 BAS1 5'-CACGAGATCTCCGACGTCGGTGTGTTTGAATCGTGTAGC-3'
RO-118 ARS1 5'-CCTATTTCTTAGCATTTTTGACGAAATTTGCT-3'
RO-119 ARS1 5'-AGCAAATTTCGTCAAAAATGCTAAGAAATAGG-3'
RO-120 ARS1 mut. 5'-CCTATTTCTTAGCATTTTTGGTGAAATTTGCT-3'
RO-121 ARS1 mut. 5'-AGCAAATTTCACCAAAAATGCTAAGAAATAGG-3'
RO-136 Vector 5'-GGCTCGAAGATCTGCC-3'
FZP20 III and IV 5'-CGTCCTCGAGCGCATATTCATTATTGCTGGGAGATCCAGGAC-3'
FZP21 III and IV 5'-CGTCCTCGAGCGCATATTCACGGGTCCTGGGAGATCCAGGAC-3'

Plasmid pR116 carries an ADE5-lacZ fusion and was constructed by first introducing a 3.0-kb BamHI fragment carrying the E. coli lacZ gene (27) into the BamHI site located in the ADE5,7 gene of plasmid pYEADE5,7(5.2R) (23) to produce plasmid pR111. The ~8-kb SalI to BspEI fragment containing the ADE5-lacZ fusion from pR111 was subcloned into the SalI and XmaI sites of a modified form of pRS316, lacking the alpha -fragment of lacZ, to produce pR116.

Plasmid pR173 contains a fusion of the bacterial glutathione S-transferase (GST) gene with the full-length coding sequence of BAS1 and was constructed in three steps. First, an oligonucleotide duplex formed between RO-97 and RO-98 was inserted at the BamHI site of pGEX-5X-3 (Pharmacia Biotech Inc.) to yield pR171. The 5' end of the gene was generated by PCR using oligonucleotides RO-107 to RO-108 and pCB286 (2). The PCR fragment was cleaved with NdeI and BglII and ligated into the NdeI and BamHI sites of pR171 to yield pR172. The remainder of the BAS1 gene was cloned as a 3.5-kb BamHI fragment from pCB286, ligated into the same site in pR172 to yield pR173.

Plasmid pR175, encoding the GST-BAS2 fusion, was constructed in two steps. First, an oligonucleotide duplex formed between RO-103 and RO-104 was inserted at the BamHI and EcoRI sites of pGEX-5X-3 to yield pR174. Plasmid pCB841 (2), which carries BAS2, was partially digested with EcoRI to generate a 2.5-kb fragment that was ligated into the EcoRI site of pR174 to yield pR175. Plasmid M2025 carrying the (His)6-BAS2 fusion was a gift of D. Stillman (15).

beta -Galactosidase Assays

Transformants to be assayed for beta -galactosidase activities were inoculated in 5 ml of SD medium supplemented with 0.5 mM arginine, 0.3 mM histidine and 0.15 mM adenine and cultured for ~42 h. Each saturated culture was diluted 1:50 in 25 ml of fresh medium, with and without adenine supplementation, and grown for 5 h with shaking at 30 °C. Cells were harvested by centrifugation and frozen at -20 °C overnight. beta -Galactosidase assays were performed using whole-cell extracts (22).

Oligonucleotide Probes for Gel Mobility Shift Assays of DNA-binding Proteins

To generate labeled ADE5,7 DNA fragments, oligonucleotides RO-41 and RO-136 were end-labeled with polynucleotide kinase and [gamma -32P]ATP and were separated from unincorporated label by passage over a G25 spin column. PCR reactions were performed as described above using the labeled oligonucleotides as primers and plasmids containing the appropriate ADE5,7 sequences as templates to amplify the region between -211 and -145 (relative to the ADE5,7 start codon). The templates were pR224, carrying the wild-type ADE5,7 fragment, or selected plasmids carrying mutated ADE5,7 promoter fragments, constructs numbered 5, 7, 10, 11, 13, 14, 19, 20, 27, 29, 34, 35, 39, and 43 as listed in Table IV. After PCR, a portion of each sample was separated by electrophoresis, and concentrations of the PCR products were estimated by ethidium bromide staining in comparison with known concentrations of duplex oligonucleotides of approximately the same length.

Table IV. Effects of clustered substitutions on UASADE5,7 function

Strain AY854 (GCN4, BAS1, and BAS2) was transformed with each of the constructs listed and assayed for beta -galactosidase as described for Table II. Expression from each construct was assayed in duplicate on at least two independent transformants, and the values shown have standard errors less than 30%. Plasmid pCM81 is the empty vector and pR224 is the parental wild-type construct containing ADE5,7 sequences -211 to -148 inserted between the BglII and XhoI sites in pCM81. The remainder of the constructs are identical to pR224 except for the mutations listed in column 4 ("Mutant sequence"). The wild-type sequence for each cluster is listed in column 3 in the row with the first mutation in that cluster. Column 5 lists the number of changes necessary to convert the wild-type sequence to the mutant one. The boldface type corresponds to the regions discussed in the text: region I, clusters 2-6; region II, clusters 10-13; region III, clusters 15-16; region IV, clusters 18-20. The 5'-most nucleotide in each cluster is as follows: cluster 1, -211; cluster 2, -208; cluster 3, -205; cluster 4, -202; cluster 5, -199; cluster 6, -196; cluster 7, -193; cluster 8, -190; cluster 9, -187; cluster 10, -184; cluster 11, -181; cluster 12, -178; cluster 13, -175; cluster 14, -172; cluster 15, -169; cluster 16, -166; cluster 17, -163; cluster 18, -160; cluster 19, -157; cluster 20, -154, cluster 21, -151; cluster 22, -148. NA, not applicable. Strain AY854 (GCN4, BAS1, and BAS2) was transformed with each of the constructs listed and assayed for beta -galactosidase as described for Table II. Expression from each construct was assayed in duplicate on at least two independent transformants, and the values shown have standard errors less than 30%. Plasmid pCM81 is the empty vector and pR224 is the parental wild-type construct containing ADE5,7 sequences -211 to -148 inserted between the BglII and XhoI sites in pCM81. The remainder of the constructs are identical to pR224 except for the mutations listed in column 4 ("Mutant sequence"). The wild-type sequence for each cluster is listed in column 3 in the row with the first mutation in that cluster. Column 5 lists the number of changes necessary to convert the wild-type sequence to the mutant one. The boldface type corresponds to the regions discussed in the text: region I, clusters 2-6; region II, clusters 10-13; region III, clusters 15-16; region IV, clusters 18-20. The 5'-most nucleotide in each cluster is as follows: cluster 1, -211; cluster 2, -208; cluster 3, -205; cluster 4, -202; cluster 5, -199; cluster 6, -196; cluster 7, -193; cluster 8, -190; cluster 9, -187; cluster 10, -184; cluster 11, -181; cluster 12, -178; cluster 13, -175; cluster 14, -172; cluster 15, -169; cluster 16, -166; cluster 17, -163; cluster 18, -160; cluster 19, -157; cluster 20, -154, cluster 21, -151; cluster 22, -148. NA, not applicable.
Plasmid number Cluster number Wild-type sequence Mutant sequence Number of changes  beta -Galactosidase activity
 Derepression ratio
 -Ade +Ade
Empty vector NA NA NA NA 6.4 6.0 1.1
Wild type NA NA NA NA 120 18 6.7
1. 134-1 1 GCC CGG 3 79 16 4.9
2. 134-2 GAG 2 82 15 5.5
3. 135-1 2 CCG AAG 2 4.7 3.1 1.5
4. 28-10 GAG 3 7.4 3.2 2.3
5. 135-5 GGA 3 7.0 3.2 2.2
6. 135-2 CGG 1 16 4.9 3.3
7. 29-1 3 TCG GTG 2 6.3 3.3 1.9
8. 29-7 AGG 2 8.6 3.1 2.8
9. 136-2 GAT 3 5.9 2.8 2.1
10. 137-1 4 GTA TGG 3 59 13 4.5
11. 137-2 GCG 2 20 21 0.9
12. 138-1 5 GTG AAT 3 9.4 3.4 2.8
13. 138-4 ACC 3 7.2 3.2 2.2
14. 31-2 CGG 2 18 6.0 3.0
15. 138-3 GAG 1 37 10 3.7
16. 138-5 TCA 3 12 4.2 2.8
17. 139-1 6 ACA GGC 3 14 4.2 3.3
18. 139-3 AAC 2 11 3.6 3.0
19. 139-4 TCT 2 14 4.4 3.2
20. 140-2 7 AGT GAC 3 98 20 4.9
21. 141-1 8 GCC GGA 2 120 18 6.7
22. 141-4 AAA 3 120 14 8.6
23. 142-2 9 GAC CTA 3 40 12 3.5
24. 142-3 GGT 2 240 24 10
25. 142-1 GGC 1 210 32 6.6
26. 143-3 10 TGA AAG 3 7.8 6.4 1.3
27. 143-2 GGA 1 16 14 1.1
28. 143-4 CGG 2 14 13 1.1
29. 144-2 11 CTC CAG 2 7.7 7.3 1.0
30. 144-1a TAT 3 6.5 7.0 0.9
31. 144-1c TTG 2 10 10 1.0
32. 145-1 12 GTG ACC 3 37 37 1.0
33. 145-2 TTC 2 48 14 3.4
34. 145-3 TCA 3 7.8 6.7 1.2
35. 146-2 13 TCC GGA 3 12 11 1.1
36. 146-1 CCC 1 120 16 7.5
37. 147-1 14 TGG CCT 3 78 15 5.2
38. 147-3 GTC 3 120 16 7.5
39. 148-1 15 TAA ATC 3 25 13 1.9
40. 148-3 TCA 1 35 19 1.8
41. 148-2b TTA 1 59 13 4.5
42. 148-5 ATG 3 40 18 2.2
43. 149-2 16 TAA CCA 2 13 6.5 2.5
44. 150-1 17 CAG CTA 2 84 11 7.6
45. 150-2 AAT 2 130 17 7.6
46. 150-4 ACA 3 120 24 5.0
47. 151-1 18 CAA GAC 2 79 16 5.2
48. 151-2 CTC 2 94 17 5.5
49. 151-3 TCA 2 52 11 4.7
50. 152-1 19 TAA CCG 3 64 33 1.9
51. 152-3 GAG 2 100 42 2.4
52. 153-2 20 TGA CCA 2 61 17 3.6
53. 155-2 21 ATA CTT 2 150 32 4.7
54. 155-3 ACC 2 100 24 4.2
55. 155-12 TTG 2 110 32 3.4
56. 155-14 TGA 2 100 29 3.4
57. 154-1 22 TGCG ACAC 4 120 17 7.1
58. 154-3 ACGA 4 220 21 10
59. 154-4 CAAC 4 81 12 6.8

Oligonucleotides RO-118 and RO-119, encoding ARS1, and RO-120 and RO-121, encoding a mutant ARS1, were prepared by end labeling with polynucleotide kinase and [gamma -32P]ATP (24), removing the unincorporated ATP on G25 spin columns, and annealing equimolar amounts of each by heating to 65 °C and cooling slowly to room temperature.

DNA fragments used as competitors were prepared as described above for the radiolabeled PCR probes, omitting the end labeling and purification steps. To generate the fragment carrying mutations in both regions III and IV, RO-136 and FZP20 were used as primers in a PCR reaction. The product of this PCR reaction was then used as template in another round of PCR using RO-136 and FZP21 as primers. The PCR fragment was inserted into pCM81 and subjected to sequence analysis to verify the correct sequence.

Electrophoretic Mobility Shift Assays of DNA Binding by ABF1

Yeast strain AY862 was grown to midlog phase and harvested by centrifugation. Cells were broken using glass beads in buffer A (25 mM Hepes, pH 7.7, 50 mM KCl, 10% glycerol, and 0.5 mM EDTA). Cell lysates were clarified by centrifugation at 20,000 × g. Labeled DNA probes for electrophoretic mobility shift assays (EMSAs) were either a 32-base duplex oligonucleotide containing the ABF1 binding site from ARS1 or radiolabeled PCR fragments containing ADE5,7 sequences, prepared as described above. DNA binding assays were performed in 20 µl with 10 µg of protein in a whole cell lysate and 10 fmol of DNA in a buffer containing 25 mM Hepes, pH 7.7, 150 mM KCl, 5 mM MgCl2, 1 µg of poly(dI·dC), and 1 µg of sonicated calf thymus DNA. Binding reactions were incubated at room temperature for 30 min and separated by gel electrophoresis using 6% polyacylamide gels in 22.3 mM Tris borate, pH 8.3, 0.5 mM EDTA. The gel was pre-electrophoresed for 15 min prior to loading samples. Supershift experiments were performed by adding either preimmune serum or immune serum raised against partially purified ABF1 to the binding reactions. The antisera, kindly provided by Bruce Stillman (28), were diluted in buffer A as indicated in the legend to Fig. 3. Competitions with unlabeled DNA fragments were performed using wild-type or mutant ARS1-containing oligonucleotides, or with PCR-derived fragments of the ADE5,7 promoter prepared as described above.

Fig. 3. In vitro binding of ABF1 protein in yeast extracts to UASADE5,7. A, binding of ABF1 in whole cell extracts to radiolabeled oligonucleotide probes was analyzed by EMSA. Lanes 1-4 show the results obtained with a probe containing the ABF1 binding site in ARS1; for lanes 5-8, the probe contained nucleotide positions -211 to -145 from the ADE5,7 promoter. For lanes 1 and 5, no yeast extract was added; for all others, 10 µg of extract from strain AY862 was added. For lanes 3 and 7, a 100-fold molar excess of the unlabeled ARS1 oligonucleotide was used as competitor; for lanes 4 and 8, the same amount of a mutant unlabeled ARS1 oligonucleotide was used as competitor. The mutation in the ARS1 fragment is a two-nucleotide substitution, replacing AGCATTTTTGACG with AGCATTTTTGGTG (underlines indicate the changes from the ABF1 consensus), shown previously to abolish ABF1 binding (33). B, formation of a supershifted complex with ABF1 antibodies. ABF1-DNA complexes were formed using the labeled ADE5,7 probe as described in A, and no serum was added to the reaction (lane 2), 1 µl of a 1:10 dilution of preimmune serum was added (lane 3), or 1 µl of a 1:100 dilution or of a 1:10 dilution of a polyclonal serum prepared against partially purified ABF1 (28) was added (lanes 4 and 5, respectively), prior to electrophoresis. C, binding of ABF1 to wild-type and mutant fragments from ADE5,7. Labeled oligonucleotides identical to the ADE5,7 probe describe above (lanes 1 and 9) or containing point mutations in the clusters indicated along the top were employed in EMSAs as described above for A. The mutations in clusters 2, 3, 5, and 6 alter conserved positions in the predicted ABF1 binding site and correspond to the mutations introduced in constructs shown as numbers 5, 7, 13, 14, and 19 in Table IV. The mutations in cluster 4 (corresponding to constructs 10 and 11 in Table IV) alter only nonconserved positions in the ABF1 site. Cluster 7 lies outside the ABF1 binding site, and the mutations analyzed at these positions had no effect on UASADE5,7 function (construct 20 in Table IV). The mutations analyzed in clusters 10-13, 15, and 16 led to reduced UASADE5,7 function but altered clusters outside of the ABF1 binding site constructs 27, 29, 34, 35, 39, and 43 in Table IV).
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Electrophoretic Mobility Shift Assays of DNA Binding by BAS1

Bacterial strains containing the GST-BAS1 fusion construct or the pGEX empty vector were inoculated at a 1:100 dilution from saturated cultures into 50 ml of LB containing ampicillin. Cultures were grown for 2.5 h, and IPTG was added to 0.5 mM for induction of the GST proteins. Cells were harvested by centrifugation after an additional 2.5 h of growth, frozen in a dry ice/ethanol bath, and stored at -80 °C. Pellets were thawed on ice and resuspended in buffer A containing 10 mM 2-mercaptoethanol and a mixture of protease inhibitors: 1 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml chymostatin, and 1 mM phenylmethylsulphonyl fluoride. Cell extracts were prepared by sonication with four 15-s pulses in an ice water bath and by three cycles of freezing in a dry ice/ethanol bath and thawing in water. Cellular debris was removed by centrifugation at 20,000 × g for 15 min. Extracts were loaded onto a glutathione S-Sepharose column with a 2-ml bed volume equilibrated in buffer A. GST control and GST-BAS1 fusion proteins were eluted from the column with 10 mM reduced glutathione in buffer A. Electrophoretic mobility shift assays were performed in 10 µl using 0.25 µg of purified protein and 10 fmol of wild-type or mutant DNA fragments in a buffer containing 25 mM Hepes, pH 7.7, 50 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, 0.5 µg of poly(dI·dC), and 10% glycerol (8). Unlabeled DNA fragments were added as competitors in a 200-fold molar excess. Samples were fractionated by electrophoresis on 6% polyacrylamide gels, as described above for ABF1 binding.

Electrophoretic Mobility Shift Assays of DNA Binding by BAS2

GST-BAS2 was prepared and used in EMSAs as described above for GST-BAS1. The (His)6-BAS2 protein was prepared for use in EMSA experiments as described (13, 15) with the following modification. Crude cell lysates (1.5 mg) from bacteria transformed with GST-BAS2 plasmid pM2025 (13) or the empty vector were prepared and added to 200 µl of Ni2+ charged His-Bind resin (Novagen). The fraction that eluted with 300 mM imidazole was dialyzed against buffer: 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 100 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, and the protease inhibitors described above. Bovine serum albumin as a carrier protein was added to a final concentration of 0.75 mg/ml before storage at -80 °C. Electrophoretic mobility shift assays were performed using 1.6 µl of these purified protein fractions.

RESULTS

Transcription of ADE5,7 and Its Repression by Adenine Both Depend on BAS1 and BAS2

We began our study of adenine regulation by analyzing expression of an ADE5,7-lacZ fusion in various strains grown in the presence and absence of exogenous adenine. We assayed wild-type and mutant strains lacking BAS1, BAS2, or GCN4. We found that ADE5,7 promoter activity under derepressing conditions (without Ade medium) and repressing conditions (with Ade medium) was lost in strains containing the mutant alleles bas1-2 or bas2-2 but was not significantly affected in a strain containing the mutant gcn4 allele (Table II). These results indicated that BAS1 and BAS2 are both essential for transcriptional activation of ADE5,7 under derepressing conditions, and they are consistent with the idea that the adenine repression involves down-regulation of BAS1 or BAS2 (3). In addition, they show that GCN4 is dispensable for ADE5,7 promoter activity and its regulation by adenine under these growth conditions (7).

Table II. Effects of gcn4, bas1, and bas2 mutations on expression and adenine regulation of an ADE5,7-lacZ fusion

Yeast strains were transformed with plasmid pR116 containing an ADE5,7-lacZ fusion on a URA3 CEN plasmid and were grown in SD medium with and without adenine (see "Experimental Procedures"). The bas1-2 and bas2-2 deletion alleles have been described (2). Extracts prepared from each transformant were assayed for beta -galactosidase activity. The specific activity of beta -galactosidase is expressed as nmol of o-nitrophenyl-beta -D-galactopyranoside hydrolyzed per min per mg of protein. Values shown are averages of the results obtained from two cultures assayed in triplicate, and the S.D. values are less than 30%. -Fold repression was calculated as the expression under derepressing conditions divided by that under repressing conditions. Yeast strains were transformed with plasmid pR116 containing an ADE5,7-lacZ fusion on a URA3 CEN plasmid and were grown in SD medium with and without adenine (see "Experimental Procedures"). The bas1-2 and bas2-2 deletion alleles have been described (2). Extracts prepared from each transformant were assayed for beta -galactosidase activity. The specific activity of beta -galactosidase is expressed as nmol of o-nitrophenyl-beta -D-galactopyranoside hydrolyzed per min per mg of protein. Values shown are averages of the results obtained from two cultures assayed in triplicate, and the S.D. values are less than 30%. -Fold repression was calculated as the expression under derepressing conditions divided by that under repressing conditions.
Strain Relevant genotype  beta -Galactosidase
 Repression
+ Ade  - Ade
units -fold
AY854 GCN4 BAS1 BAS2 8.5 72 8.5
AY856 GCN4 bas1-2 BAS2 5.7 7.3 1.3
AY858 GCN4 BAS1 bas2-2 5.7 7.5 1.3
AY860 GCN4 bas1-2 bas2-2 7.3 8.5 1.2
AY957 gcn4 BAS1 BAS2 13 96 7.4
AY862 gcn4 bas1-2 bas2-2 7.1 4.9 0.7

Identification of a Minimal ADE5,7 Promoter Fragment with UAS Function

The results in Table II suggest that the ability of BAS1 and BAS2 to activate ADE5,7 transcription is completely inhibited in adenine-replete cells. It was not known, however, whether ADE5,7 promoter activity is dependent on any additional positive regulators that might be subject to adenine repression or whether repression involves a DNA binding repressor. To address these issues, we set out to identify the cis-acting sequences required for adenine-regulated promoter activity by determining the smallest DNA fragment from ADE5,7 sufficient to confer adenine-repressible UAS function on a CYC1-lacZ reporter (21). Various fragments of the ADE5,7 promoter were generated by PCR and inserted ~185 nucleotides upstream of the CYC1 transcription initiation sites where the UASCYC normally resides (Fig. 1). These constructs were introduced into four different yeast strains carrying wild-type or mutated alleles of GCN4, BAS1, and BAS2, and beta -galactosidase expression was assayed after growing transformants in minimal medium containing or lacking adenine. As expected, expression of beta -galactosidase in transformants of each strain bearing the parental CYC1-lacZ construct lacking UASCYC1 was very low (~10 units) and essentially unaffected by adenine supplementation or mutations in any of the regulatory genes (Table III, line 19). The ADE5,7 promoter fragments exhibited a wide range of expression from the CYC1 promoter in the wild-type strain, and in many cases promoter activity was repressed by adenine in the medium. As seen with the authentic ADE5,7 promoter (Table II), expression depended upon the wild-type alleles of BAS1 and BAS2 but not upon GCN4 (Table III). In addition to these adenine-regulated constructs, promoter fragments with 3' end points at positions -183 (Table III, lines 3 and 7) or at -160 (Table III, lines 2 and 10) exhibited significant activity that was independent of GCN4, BAS1, and BAS2 and was largely unaffected by adenine in the medium.

Fig. 1. Deletion and substitution mutations defining the UASADE5,7. The top section shows a schematic of the 139-nucleotide fragment from the ADE5,7 promoter with nucleotide positions numbered relative to the initiation codon. Hatched and solid boxes represent the distal and proximal BAS1 binding sites, respectively, that contain the core sequence TGACTC (3). The distal site is located between -217 and -212, and the proximal site is located between -184 and -179. A, constructs used in the deletion analysis. PCR fragments with 5' end points at positions -271, -233, -211, or -195 and 3' end points at -183, -160, -145, or -133 were generated using oligonucleotides listed in Table I and are indicated by the lines shown below the schematic. These fragments were inserted in place of UASCYC1 in plasmid pLG669ZDelta XhoI, and the resulting constructs were introduced into yeast strain AY854 (wild type). beta -Galactosidase activities in whole cell extracts were determined after growing the transformants under derepressing (-Ade) or repressing conditions (+Ade) and are listed in Table III. These results have been corrected for UAS activity that is not attributed to the three transcriptional activators GCN4, BAS1, or BAS2 by subtracting the activity found in strain AY862 (gcn4 bas1 bas2) and are shown to the right of each construct. In cases where the corrected value was a negative number, it is enclosed by parentheses. NA, not applicable. B, constructs used to analyze mutations in the BAS1 binding site. A point mutation from TGACTC to TGAATTC in the core sequence is represented by an X.
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Table III. Analysis of UASADE5,7 function conferred on a CYC1-lacZ fusion by different fragments from the ADE5,7 promoter

Plasmids containing either no insert (line 19, vector pLG699ZDelta XhoI) or containing various fragments from the ADE5,7 promoter (lines 1-18, plasmids pR133 to pR152) were introduced into strains AY854 (GCN4 BAS1 BAS2), AY860 (GCN4 bas1-2 bas2-2), AY957 (gcn4 BAS1 BAS2), and AY862 (gcn4 bas1-2 bas2-2), and the transformants were assayed for beta -galactosidase activities after growth under repressing (+Ade) and derepressing (-Ade) conditions, as described in Table II. Values shown are averages of the results obtained from 2-4 cultures assayed in triplicate, and the S.D. values are less than 30%. The nucleotide positions of the ends of the ADE5,7 fragments that are inserted at the XhoI site of pLG699ZDelta XhoI are listed in the column labeled "ADE5,7 fragment." Plasmids containing either no insert (line 19, vector pLG699ZDelta XhoI) or containing various fragments from the ADE5,7 promoter (lines 1-18, plasmids pR133 to pR152) were introduced into strains AY854 (GCN4 BAS1 BAS2), AY860 (GCN4 bas1-2 bas2-2), AY957 (gcn4 BAS1 BAS2), and AY862 (gcn4 bas1-2 bas2-2), and the transformants were assayed for beta -galactosidase activities after growth under repressing (+Ade) and derepressing (-Ade) conditions, as described in Table II. Values shown are averages of the results obtained from 2-4 cultures assayed in triplicate, and the S.D. values are less than 30%. The nucleotide positions of the ends of the ADE5,7 fragments that are inserted at the XhoI site of pLG699ZDelta XhoI are listed in the column labeled "ADE5,7 fragment."
Plasmid ADE