Overlapping Positive and Negative GATA Factor Binding Sites Mediate Inducible DAL7 Gene Expression in Saccharomyces cerevisiae
1999; Elsevier BV; Volume: 274; Issue: 39 Linguagem: Inglês
10.1074/jbc.274.39.28026
ISSN1083-351X
AutoresRajendra Rai, Jon R. Daugherty, Thomas S. Cunningham, Terrance Cooper,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoAllantoin pathway gene expression inSaccharomyces cerevisiae responds to two different environmental stimuli. The expression of these genes is induced in the presence of allantoin or its degradative metabolites and repressed when a good nitrogen source (e.g. asparagine or glutamine) is provided. Three types of cis-acting sites and trans-acting factors are required for allantoin pathway gene transcription as follows: (i)UAS NTR element associated with the transcriptional activators Gln3p and Gat1p, (ii)URS GATA element associated with the repressor Dal80p, and (iii) UIS ALL element associated with the Dal82 and Dal81 proteins required for inducer-dependent transcription. Most of the work leading to the above conclusions has employed inducer-independent allantoin pathway genes (e.g. DAL5 and DAL3). The purpose of this work is to extend our understanding of these elements and their roles to inducible allantoin pathway genes using theDAL7 (encoding malate synthase) as a model. We show that eight distinct cis-acting sites participate in the process as follows: a newly identified GC-rich element, two UAS NTR, two UIS ALL, and threeURS GATA elements. The two GATA-containingUAS NTR elements are coincident with two of the three GATA sequences that make up the URS GATAelements. The remaining URS GATA GATA sequence, however, is not a UAS NTR element but appears to function only in repression. The data provide insights into how these cis- and trans-acting factors function together to accomplish the regulated expression of the DAL7 gene that is observedin vivo. Allantoin pathway gene expression inSaccharomyces cerevisiae responds to two different environmental stimuli. The expression of these genes is induced in the presence of allantoin or its degradative metabolites and repressed when a good nitrogen source (e.g. asparagine or glutamine) is provided. Three types of cis-acting sites and trans-acting factors are required for allantoin pathway gene transcription as follows: (i)UAS NTR element associated with the transcriptional activators Gln3p and Gat1p, (ii)URS GATA element associated with the repressor Dal80p, and (iii) UIS ALL element associated with the Dal82 and Dal81 proteins required for inducer-dependent transcription. Most of the work leading to the above conclusions has employed inducer-independent allantoin pathway genes (e.g. DAL5 and DAL3). The purpose of this work is to extend our understanding of these elements and their roles to inducible allantoin pathway genes using theDAL7 (encoding malate synthase) as a model. We show that eight distinct cis-acting sites participate in the process as follows: a newly identified GC-rich element, two UAS NTR, two UIS ALL, and threeURS GATA elements. The two GATA-containingUAS NTR elements are coincident with two of the three GATA sequences that make up the URS GATAelements. The remaining URS GATA GATA sequence, however, is not a UAS NTR element but appears to function only in repression. The data provide insights into how these cis- and trans-acting factors function together to accomplish the regulated expression of the DAL7 gene that is observedin vivo. Inducible nitrogen catabolic genes in Saccharomyces cerevisiae respond to at least two different types of signals. One is a dominant, general signal that monitors the overall nitrogen supply of the cell. The availability of readily transported and metabolized nitrogen sources result in high nitrogen catabolite repression (NCR) 1The abbreviations used are:NCRnitrogen catabolite repressionEMSAelectrophoretic mobility shift assaybpbase pairPCRpolymerase chain reaction (1Cooper T.G. Strathern J.N. Jones E.W. Broach J. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar, 2Wiame J.-M. Grenson M. Arst H. Adv. Microb. Physiol. 1985; 26: 1-87Crossref PubMed Scopus (239) Google Scholar). Alternatively, low concentrations of good nitrogen sources or the presence of only slowly transported and/or metabolized nitrogen sources in the environment of cells results in low NCR. Genes encoding proteins involved in the transport and metabolism of poor nitrogen sources are NCR-responsive, i.e. they are expressed at low levels under conditions of high NCR and at higher levels under conditions of low NCR; the latter is also referred to occasionally as derepression (1Cooper T.G. Strathern J.N. Jones E.W. Broach J. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 39-99Google Scholar,2Wiame J.-M. Grenson M. Arst H. Adv. Microb. Physiol. 1985; 26: 1-87Crossref PubMed Scopus (239) Google Scholar). The second signal is pathway-specific and derives from the presence of a particular nitrogen source (e.g. allantoin or one of its metabolites) (3Cooper T.G. Lawther R.P. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2340-2344Crossref PubMed Scopus (21) Google Scholar). Responses to these two physiological signals are integrated at transcription resulting in fine regulation of catabolic gene expression that ranges from high to low as needed to exploit most effectively the prevailing environmental nitrogen supplies for the needs of the cell. nitrogen catabolite repression electrophoretic mobility shift assay base pair polymerase chain reaction Allantoin catabolic pathway gene expression has been a useful model with which to study the nature of nitrogen regulatory signals and the cell's detection of and response to them (see Ref. 4Cooper T.G. Brambl R. Marzluf G. Mycota. Springer-Verlag, Berlin1996: 139-169Google Scholar for a comprehensive review of the allantoin pathway literature; shorter literature reviews covering contributions from the range of investigators in the field of yeast GATA factors and nitrogen regulation per se appear in the introductions of Refs. 5Coffman J.A. Rai R. Loprete D.M. Cunningham T. Svetlov V. Cooper T.G. J. Bacteriol. 1997; 179: 3416-3429Crossref PubMed Google Scholar and6Svetlov V. Cooper T.G. J. Bacteriol. 1997; 179: 7644-7652Crossref PubMed Google Scholar). These studies identified three types of cis-acting elements and trans-acting factors associated with allantoin pathway genes (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar). The cis-acting element mediating NCR-sensitive transcriptional activation is UAS NTR (UASNitrogen-Regulated) (9Miller S.M. Magasanik B. Mol. Cell. Biol. 1991; 11: 6229-6247Crossref PubMed Scopus (42) Google Scholar,10Bysani N. Daugherty J.R. Cooper T.G. J. Bacteriol. 1991; 173: 4977-4982Crossref PubMed Google Scholar), consisting of two separated dodecanucleotides each with the sequence GATAA at its core (10Bysani N. Daugherty J.R. Cooper T.G. J. Bacteriol. 1991; 173: 4977-4982Crossref PubMed Google Scholar). UAS NTR has been shown to be both necessary and sufficient for NCR-sensitiveDAL gene transcription (11Cooper T.G. Rai R. Yoo H.-S. Mol. Cell. Biol. 1989; 9: 5440-5444Crossref PubMed Scopus (34) Google Scholar); Gln3p and Gat1p are required for this transcription (12Coffman J.A. Rai R. Cunningham T. Svetlov V. Cooper T.G. Mol. Cell. Biol. 1996; 16: 847-858Crossref PubMed Scopus (121) Google Scholar). The Magasanik group (13Mitchell A.P. Magasanik B. Mol. Cell. Biol. 1984; 4: 2758-2766Crossref PubMed Scopus (97) Google Scholar, 14Minehart P.L. Magasanik B. Mol. Cell. Biol. 1991; 11: 6216-6228Crossref PubMed Scopus (154) Google Scholar) reported that antibody against a Gln3p peptide containing the GATA family zinc finger motif precipitated a synthetic DNA fragment containing seven repeats of a 32-bp GLN1 promoter fragment containing a GATA sequence from crude extract; this led them to suggest that Gln3p bound to GATA sequences. Their observation was consistent with the finding that the deduced Gln3p sequence contains a zinc finger motif homologous to the mammalian GATA-binding family of transcription factors (15Omichinski J.G. Close G.M. Schaad O. Felsenfeld G. Trainor C. Appella E. Stahl S.J. Gronenborn A.M. Science. 1993; 261: 438-446Crossref PubMed Scopus (407) Google Scholar). By using electrophoretic mobility shift assays (EMSAs), direct binding of Gln3p to UAS NTR sequences has been demonstrated (16Cunningham T.S. Svetlov V. Rai R. Smart W. Cooper T.G. J. Bacteriol. 1996; 178: 3470-3479Crossref PubMed Google Scholar,17Blinder D. Magasanik B. J. Bacteriol. 1995; 177: 4190-4193Crossref PubMed Google Scholar). Multiple UAS NTR homologous sequences are situated upstream of all allantoin pathway genes, and these sequences have been shown to account for NCR-sensitive, Gln3p-dependent expression of the inducer-independentDAL5 and DAL3 genes (8Rai R. Genbauffe F.S. Sumrada R.A. Cooper T.G. Mol. Cell. Biol. 1989; 9: 602-608Crossref PubMed Scopus (49) Google Scholar, 10Bysani N. Daugherty J.R. Cooper T.G. J. Bacteriol. 1991; 173: 4977-4982Crossref PubMed Google Scholar, 11Cooper T.G. Rai R. Yoo H.-S. Mol. Cell. Biol. 1989; 9: 5440-5444Crossref PubMed Scopus (34) Google Scholar, 18Cunningham T.S. Dorrington R.A. Cooper T.G. J. Bacteriol. 1994; 176: 4718-4725Crossref PubMed Google Scholar, 38Rai R. Genbauffe F.S. Cooper T.G. J. Bacteriol. 1987; 169: 3521-3524Crossref PubMed Google Scholar). The cis-acting element mediating inducer responsiveness of the allantoin pathway genes is the dodecanucleotide element,UIS ALL (UpstreamInduction Sequence) (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar, 19Van Vuuren H.J.J. Daugherty J.R. Rai R. Cooper T.G. J. Bacteriol. 1991; 173: 7186-7195Crossref PubMed Scopus (28) Google Scholar). The inducer to which proteins associated with the UIS ALLrespond is the last intermediate of the allantoin pathway, allophanate, or its non-metabolized analogue, oxalurate (20Sumrada R. Cooper T.G. J. Bacteriol. 1974; 117: 1240-1247Crossref PubMed Google Scholar). The allophanate-inducible DAL genes contain one or two copies ofUIS ALL (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar, 21Dorrington R.A. Cooper T.G. Nucleic Acids Res. 1993; 21: 3777-3784Crossref PubMed Scopus (29) Google Scholar). The DAL81/DURL/UGA35and DAL82/DURM gene products are required for this inducer responsiveness (22Jacobs E. Dubois E. Hennaut C. Wiame J.-M. Curr. Genet. 1981; 4: 13-18Crossref PubMed Scopus (19) Google Scholar, 23Jacobs E. Dubois E. Wiame J.-M. Curr. Genet. 1985; 9: 333-339Crossref PubMed Scopus (7) Google Scholar, 24Coornaert D. Vissers S. Andre B. Gene ( Amst. ). 1991; 97: 163-171Crossref PubMed Scopus (48) Google Scholar, 25Turoscy V. Cooper T.G. J. Bacteriol. 1982; 151: 1237-1246Crossref PubMed Google Scholar). Dal82p has been shown to be theUIS ALL DNA-binding protein whose binding to DNA is independent of inducer (21Dorrington R.A. Cooper T.G. Nucleic Acids Res. 1993; 21: 3777-3784Crossref PubMed Scopus (29) Google Scholar). Whereas Dal82p appears to be a pathway-specific regulatory element, Dal81p functions more broadly, being required for induced expression of the UGA(γ-aminobutyrate catabolic pathway) and AGP1 (tryptophan uptake) genes as well as those of the allantoin pathway (26Vissers S. Andre B. Muyldermans F. Grenson M. Eur. J. Biochem. 1990; 187: 611-616Crossref PubMed Scopus (42) Google Scholar, 27Iraqui I. Vissers S. Bernard F. Ol de Craene J. Boles E. Urrestarazu A. Andre B. Mol. Cell. Biol. 1999; 19: 989-1001Crossref PubMed Google Scholar). A third type of cis-acting element and cognate transcription factor down-regulate DAL gene expression; they are URSGATA and Dal80p, respectively (28Chisholm G. Cooper T.G. Mol. Cell. Biol. 1982; 2: 1088-1095Crossref PubMed Scopus (36) Google Scholar, 29Cunningham T.S. Cooper T.G. J. Bacteriol. 1993; 175: 5851-5861Crossref PubMed Google Scholar). TheDAL80 locus was first identified genetically (28Chisholm G. Cooper T.G. Mol. Cell. Biol. 1982; 2: 1088-1095Crossref PubMed Scopus (36) Google Scholar). Mutations in this gene increase allantoin pathway-inducible gene expression in the absence of inducer to the same level observed in a wild type strain grown with inducer. This observation led to the suggestion that Dal80p functions to reduce inducible gene expression when inducer is absent (28Chisholm G. Cooper T.G. Mol. Cell. Biol. 1982; 2: 1088-1095Crossref PubMed Scopus (36) Google Scholar). Dal80p was subsequently found to perform a similar function for the inducible UGA genes (33Vissers S. Andre B. Muyldermans F. Grenson M. Eur. J. Biochem. 1989; 181: 357-361Crossref PubMed Scopus (53) Google Scholar, 34Coornaert D. Vissers S. Andre B. Grenson M. Curr. Genet. 1992; 21: 301-307Crossref PubMed Scopus (49) Google Scholar). However, Dal80p also down-regulates inducer-independent DAL gene expression 2–20-fold (18Cunningham T.S. Dorrington R.A. Cooper T.G. J. Bacteriol. 1994; 176: 4718-4725Crossref PubMed Google Scholar), suggesting a more general physiological function than simply maintaining inducible gene expression at low levels. Gln3p and Dal80p were proposed to be opposing regulators of most NCR-sensitive nitrogen catabolic genes (30Daugherty J.R. Rai R. ElBerry H.M. Cooper T.G. J. Bacteriol. 1993; 175: 64-73Crossref PubMed Google Scholar). This proposal has been subsequently supported by data from other laboratories (31Andre B. Talibi D. Boudekou S.S. Hein C. Vissers S. Coornaert D. Nucleic Acids Res. 1995; 23: 558-564Crossref PubMed Scopus (46) Google Scholar). The deduced amino acid sequence of Dal80p contains a zinc finger motif homologous to the one in Gln3p (32Cunningham T.S. Cooper T.G. Mol. Cell. Biol. 1991; 11: 6205-6215Crossref PubMed Scopus (122) Google Scholar, 34Coornaert D. Vissers S. Andre B. Grenson M. Curr. Genet. 1992; 21: 301-307Crossref PubMed Scopus (49) Google Scholar). Prompted to test the implication of this homology, we demonstrated Dal80p to be a DNA-binding protein whose optimal binding site, URS GATA, consists of two GATAAG-containing sequences separated 15–35 bp, oriented tail-to-tail or head-to-tail (29Cunningham T.S. Cooper T.G. J. Bacteriol. 1993; 175: 5851-5861Crossref PubMed Google Scholar). The requirement of two GATA-containing sequences in a Dal80p-binding site correlates with the fact that Dal80p molecules have been recently shown to dimerize via a leucine zipper motif in their C-terminal domains using two-hybrid assays (35Svetlov V.V. Cooper T.G. J. Bacteriol. 1998; 180: 5682-5688Crossref PubMed Google Scholar). The structural similarity of UAS NTR andURS GATA sites led to the suggestion that Dal80p-binding sites might be Gln3p-binding sites as well (18Cunningham T.S. Dorrington R.A. Cooper T.G. J. Bacteriol. 1994; 176: 4718-4725Crossref PubMed Google Scholar). This suggestion was supported by the demonstration that the GATA sequences of the UGA4 and DAL3 UAS NTRs that bind Gln3p also bind Dal80p (18Cunningham T.S. Dorrington R.A. Cooper T.G. J. Bacteriol. 1994; 176: 4718-4725Crossref PubMed Google Scholar). The fact that Gln3p can bind to a single GATA sequence while Dal80p cannot explains why some genes, such as DAL5, are Gln3p-dependent but largely immune from regulation by Dal80p (4Cooper T.G. Brambl R. Marzluf G. Mycota. Springer-Verlag, Berlin1996: 139-169Google Scholar). The bulk of our understanding of allantoin pathway gene regulation and the role played by Dal80p down-regulating Gln3p transcriptional activation has been derived from studies with inducer-independent genes such as DAL5 and DAL3 (4Cooper T.G. Brambl R. Marzluf G. Mycota. Springer-Verlag, Berlin1996: 139-169Google Scholar). Our objective here is to extend these studies to the inducible allantoin pathway genes whose expression is not only NCR-sensitive like that of DAL5 andDAL3, and Dal80p-regulated like that of DAL3, but also inducer-responsive. The inducible DAL7 gene is the one we investigated. The results obtained delineate which of the many DAL7 upstream sequences that are homologous to known transcription factor binding sites actually participate in DAL7 expression. They further identify a cis-acting site not previously known to mediate allantoin pathway gene transcription and suggest a crude picture of how Dal80p, Gln3p, and Dal82p might work together to regulate inducer-dependent DAL7 expression. The strains used in this work are listed in Table I. When correlating data from this and other work, it is important to recognize that the fold induction (induced level divided by the basal level of activity) for and Dal80p regulation of DAL7 expression is highly strain-dependent. It ranges from as much as 20–30-fold in strains such as RH218 (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar) to as little as 2–4-fold in the case of strains derived from Σ1278b. 2J. Daugherty and T. G. Cooper, unpublished observations). The source(s) of these differences are not known. The media used in this work were all standard formulations.Table IYeast strains used in this workStrainGenotypeΣ1278bMATαTCY1MATα lys2 ura3TCY17MATα lys2 ura3 dal80::hisG-URA3-hisGM1682–19bMATα ura3–52 trp1-289 Open table in a new tab Transformation of S. cerevisiae and the procedures used to assay β-galactosidase have been described earlier (36Smart W.C. Coffman J.A. Cooper T.G. Mol. Cell. Biol. 1996; 16: 5876-5887Crossref PubMed Scopus (29) Google Scholar). The cultures were grown (2% glucose, 0.17% YNB (Difco), and 0.1% of the indicated nitrogen source) for 16–24 h to a cell density ofA 600 nm = 0.4 to 0.8 as measured on a Zeiss, Gilford, or Spectronic Genesys 5 spectrophotometer. After the desired cell density was reached, 10-ml samples were harvested and assayed (36Smart W.C. Coffman J.A. Cooper T.G. Mol. Cell. Biol. 1996; 16: 5876-5887Crossref PubMed Scopus (29) Google Scholar). Statistics of the β-galactosidase measurements and their associated error (normally 5–10%) have been analyzed in detail (36Smart W.C. Coffman J.A. Cooper T.G. Mol. Cell. Biol. 1996; 16: 5876-5887Crossref PubMed Scopus (29) Google Scholar). In this regard, it is important to emphasize that data (absolute values) in Fig. 2 cannot be quantitatively compared with those in Figs. 3 or 4even though some of the same plasmids were used. This limitation derives from the use of different spectrophotometers and different strains; the spectrophotometers mentioned above, as is true for all spectrophotometers, differ significantly in their light scattering characteristics. These differences can range up to 2-fold depending upon the density of the sample. Although it is inappropriate to compare the absolute values between separate figures, critical comparisons of values contained within a figure and the patterns of regulation observed from one experiment (figure) to another are justified (36Smart W.C. Coffman J.A. Cooper T.G. Mol. Cell. Biol. 1996; 16: 5876-5887Crossref PubMed Scopus (29) Google Scholar).Figure 3Expression of wild type and mutantDAL7 promoter fragments (synthetic oligonucleotides) cloned into heterologous expression vector plasmid pNG15. Wild type sequences of the various cis-acting elements appear ascapital letters in the figure and those of the mutant alleles in lowercase letters. Transformants of parent strain M1682-19b was grown and β-galactosidase assayed as described in Fig.2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Expression of wild type and mutantDAL7 promoter fragments described in Fig. 3 in wild type (TCY1) and gln3Δ (RR91) strains growing in minimal YNB medium containing 2% glucose and 0.1% γ-aminobutyric acid (GABA) as sole carbon and nitrogen sources. Plasmids and assay conditions were as described in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The heterologous expression plasmids used in Figs. Figure 2, Figure 3, Figure 4 were constructed using double-stranded, synthetic oligonucleotides (the coordinates are indicated) that were extended by the addition of five bases of the SalI and EagI restriction endonuclease sites to their 5′ and 3′ termini, respectively. These synthetic fragments were then cloned into plasmid pNG 15 (10Bysani N. Daugherty J.R. Cooper T.G. J. Bacteriol. 1991; 173: 4977-4982Crossref PubMed Google Scholar) digested with the same enzymes. All constructs were sequenced before being used. The native DAL7 promoter fragments used in Figs. 9 and 10were created using PCR-based methods and cloned into theBamHI site of plasmid pVAN2 (Fig.1) which generated plasmid pRR352, the wild type parent plasmid. Plasmid pVAN2 was derived from plasmid pHP41 (37Park H.-D. Luche R.M. Cooper T.G. Nucleic Acids Res. 1992; 20: 1909-1915Crossref PubMed Scopus (74) Google Scholar) and does not contain any of the CYC1 promoter sequences. The method of creating mutations in the promoter region involved using mutagenic primers during PCR. The heat-stable DNA polymerase used in these reactions was PWO polymerase from Roche Molecular Biochemicals. Reaction conditions were standard and described by the manufacturer. All of the PCR-generated DNA fragments were cloned into the BamHI site of plasmid pUC18. This vector was chosen to facilitate cloning and DNA sequencing of the fragments. The sequences of all of the PCR-generated DNA fragments were determined prior to being used. The mutations introduced into the promoter fragments are listed in Table II. The mutant fragments were identical to the wild type parent except at the positions listed; those positions carried the indicated substitution mutations.Figure 10Expression of wild type and promoter mutant alleles of a DAL7-lacZ fusion plasmid, containing a native DAL7 promoter. Plasmids were transformed into wild type (TCY1) and dal80 mutant (TCY17) strains. The experiment was conducted as described in Fig. 9.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1The plasmid used for cloning wild type and mutant alleles of the native DAL7 promoter fused in frame to the lacZ gene.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IICoordinates of the mutated bases in plasmids containing full-length DAL7 promoter fragments fused in frame with lacZPlasmid numberCoordinates of the mutated basesaUnderlined bases indicate those that were mutated.Mutated sequencepRR373−281 to −285,TTATCggtcapRR374−252 to −263, GAAAGTTGCGGTGAcctTTGtgtapRR375−245 to −249, GATAGtgaccpRR371−235 to −240, CCGCGGgacgtcpRR376−217 to −228, GAAAATTGCGTTGAcctTTtggaapRR380−204 to −208, TTATCggtcapRR377−171 to −175, TTATCggtcapRR372−163 to −167, GATAAtgacca Underlined bases indicate those that were mutated. Open table in a new tab EMSAs were performed as described earlier (29Cunningham T.S. Cooper T.G. J. Bacteriol. 1993; 175: 5851-5861Crossref PubMed Google Scholar) including preparation of the double-stranded oligonucleotides, Klenow-mediated fill in reactions to generate [32P]dCTP-labeled probes, preparation of the Dal80p extracts, binding reactions, and electrophoresis. All oligonucleotides contained SalI and EagI overhangs at their 5′ and 3′ ends, respectively, to provide targets for the fill-in reactions and the ability to clone the fragments. In cases where a particular GATA sequence was mutated, the wild type sequence starting with the 5′-guanosine of the GATA was changed to a MunI restriction site. For example, if the wild type sequence was 5′-GATAAG-3′ (or 5′GATAGT-3”), it was converted to the sequence 5′-CAATTG-3′. (See TableIII for the mutated bases and their coordinates in the DNA fragments used in the EMSAs.) DNA fragments DAL3–5, containing the three clustered GATAs from the DAL3gene, and DAL3–35, a synthetic fragment containing two GATA elements in tail-to-tail orientation, have been described in detail elsewhere (29Cunningham T.S. Cooper T.G. J. Bacteriol. 1993; 175: 5851-5861Crossref PubMed Google Scholar).Table IIICoordinates of DNA fragments used in the EMSAsOligonucleotideFragment end point coordinatesaThe first base and the 4-base overhang of aSalI restriction site were added to the 5′ end of each oligonucleotide. Similarly, 5 bases of an EagI site were added to the 3′ end.Coordinates of mutated GATAsDAL7–1−183 to −122DAL7–2−239 to −156DAL7–3−292 to −214DAL7–4−265 to −196DAL7–5−183 to −122−176-(CTTATC)-171 > caattgDAL7–6−183 to −122−167(GATAAG)-162 > caattgDAL7–7−183 to −122−133(GATAGT)-128 > caattgDAL7–8−239 to −156−167(GATAAG)-162 > caattgDAL7–9−239 to −156−176(CTTATC)-171 > caattgDAL7–10−239 to −156−208(CTTATC)-203 > caattgDAL7–11−196 to −148a The first base and the 4-base overhang of aSalI restriction site were added to the 5′ end of each oligonucleotide. Similarly, 5 bases of an EagI site were added to the 3′ end. Open table in a new tab Previous experiments identified two types of positively acting DAL7 elements that participate in physiologically relevant heterologous reporter gene expression,UAS NTR and UIS ALL (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar). However, these studies were unable to address adequately how these cis-acting elements and the trans-acting proteins associated with them cooperate to mediate inducer-dependent, Dalp80-regulatedDAL7 expression. The inadequacy derived, in part, from a lack of knowledge about the biochemical function of Dal80p and clear identification of which of the cis-active element homologous sequences actually participated in DAL7 expression (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar). When early 5′ deletion data (derived from a DAL7-lacZ fusion plasmid (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar)) are analyzed from the perspective of sequences homologous to those of known cis-acting elements, there are instances in which single deletions potentially removed more than one cis-acting element. The region between positions −290 and −221, relative to the ATG, is not only an area where our information is incomplete but also one in which existing deletions have the greatest effect upon expression (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar). To characterize further the regions most responsible forDAL7 expression, we cloned a synthetic fragment, containing sequences from −320 to −199, into a heterologous expression vector (plasmid pJD98, see “Experimental Procedures”); sequential 5′ deletions were then used to delineate the cis-acting elements (Fig.2). The first two deletions, to −300 and −266, had little effect upon β-galactosidase production (Fig. 2,plasmids pJD98, pJD95, and pJD68). This argues that the UAS NTR homologous sequence between positions −281 and −286 (5′-ATTATC-3′) does not demonstrably function as a UAS element in this fragment. However, deletion of the next 12 bases (to position −254) decreased reporter gene expression 3–4-fold in the presence or absence of inducer (plasmid pJD72). This region contained a sequence homologous to UIS ALLand has been shown to bind Dal82p (21Dorrington R.A. Cooper T.G. Nucleic Acids Res. 1993; 21: 3777-3784Crossref PubMed Scopus (29) Google Scholar). These results argue that this UIS ALL sequence participates in reporter gene expression. When the above data and those published earlier (7Yoo H.S. Cooper T.G. Mol. Cell. Biol. 1989; 9: 3231-3243Crossref PubMed Scopus (34) Google Scholar) are considered together, the only region of plasmid pJD68 not analyzed is between positions −244 and −230. To remedy this, we synthesized a mutant form of plasmid pJD68 in which a GC-rich sequence, 5′-CCGCGG-3′, at positions −240 to −235 was mutated. This particular sequence was chosen because it is a GC-rich inverted repeat, both characteristics of multiple transcription factor binding sites. This alteration affected reporter gene expression in two ways (Fig. 2, compare plasmids pJD68 and pJD92). First, basal and induced levels of β-galactosidase production decrease 110- and 12-fold, respectively. Second, inducer responsiveness increases 10-fold relative to the wild type; there is a 2-fold response with plasmid pJD68 and 20-fold with plasmid pJD92 (PRO+ level divided by the PRO level). We next analyzed the potential cis-acting sites in plasmid pJD68 using point mutations that did not otherwise alter the number of bases or spacing of putative elements relative to the heterologous TATA elements and mRNA start sites. As shown in Fig.3, these mutations decreased reporter gene expression in cells growing in glucose/proline medium 2–60-fold. A more modest result (13-fold maximum) was observed with glucose/asparagine medium. Mutation of the 5′UIS ALL element (plasmid pJD152) decreased reporter gene expression about 2.5-fold, which is similar to the 4-fold decrease observed in Fig. 2. When the 5′-most UASNTRhomologous sequence was mutated, lacZ expression decreased about 2-fold (plasmid pJD156). Mutation of the GC-rich element (plasmid pJD92) has already been described, but it is worth noting that of all the mutations exam
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