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• W2028004003 abstract "The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation. It is modulated by α-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of α-isopropylmalate, Leu3p is a transcriptional activator. In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor. The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS Lett. 390, 191–195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins. Here we present experimental support for this notion and address the mechanism of masking. We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain. An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator. Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for α-isopropylmalate-dependent interaction between these segments. Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd. The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking. The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation. It is modulated by α-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of α-isopropylmalate, Leu3p is a transcriptional activator. In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor. The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS Lett. 390, 191–195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins. Here we present experimental support for this notion and address the mechanism of masking. We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain. An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator. Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for α-isopropylmalate-dependent interaction between these segments. Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd. The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking. The Leu3 protein of yeast belongs to a class of transcriptional regulators whose members are characterized by a Zn(II)2-Cys6 binuclear cluster in their DNA binding domains (1Marmorstein R. Carey M. Ptashne M. Harrison S.C. Nature. 1992; 356: 408-414Crossref PubMed Scopus (553) Google Scholar, 2Marmorstein R. Harrison S.C. Genes Dev. 1994; 8: 2504-2512Crossref PubMed Scopus (149) Google Scholar). Many of them also have the ability to be modulated, i.e. to respond to metabolic signals. Well studied examples of modulation include Gal4p, which is activated (“unmasked”) by galactose in a process that involves changes in the interaction between Gal4p and an auxiliary protein known as Gal80p (3Leuther K.K. Johnston S.A. Science. 1992; 256: 1333-1335Crossref PubMed Scopus (118) Google Scholar,4Leuther K.K. Salmeron J.M. Johnston S.A. Cell. 1993; 72: 575-585Abstract Full Text PDF PubMed Scopus (105) Google Scholar), and Hap1p, the activation of which by heme is thought to be brought about by dissociation of cellular factors that allow the DNA binding domain to become functional and its activation domain (AD) 1The abbreviations used are: AD, activation domain; IPM, isopropylmalate; kbp, kilobase pair; UASL, upstream activating sequence in the promoters of Leu3p-regulated genes; WT, wild type; PCR, polymerase chain reaction; MR, middle region; DB, DNA binding domain. to become exposed (5Zhang L. Guarente L. J. Biol. Chem. 1994; 269: 14643-14647Abstract Full Text PDF PubMed Google Scholar,6Zhang L. Guarente L. EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (255) Google Scholar). Leu3p is modulated by α-IPM,1 an intermediate in leucine biosynthesis (7Hampsey D.M. Lewin A.S. Kohlhaw G.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1270-1274Crossref PubMed Scopus (16) Google Scholar). Leu3p binds to UASL elements in the promoters of genes involved in the biosynthesis of branched chain amino acids (8Friden P. Schimmel P. Mol. Cell. Biol. 1988; 8: 2690-2697Crossref PubMed Scopus (81) Google Scholar, 9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar) and, surprisingly, in ammonia assimilation in yeast (10Hu Y. Cooper T.G. Kohlhaw G.B. Mol. Cell. Biol. 1995; 15: 52-57Crossref PubMed Scopus (42) Google Scholar). The finding that the GDH1 gene is regulated by Leu3p and α-IPM (10Hu Y. Cooper T.G. Kohlhaw G.B. Mol. Cell. Biol. 1995; 15: 52-57Crossref PubMed Scopus (42) Google Scholar) led to the hypothesis that α-IPM serves as a signal molecule that communicates the status of amino acid biosynthetic activity (as represented by the leucine pathway) to more central points of nitrogen metabolism. It is of interest in this context that theLEU3 gene itself is controlled by Gcn4p (11Zhou K. Brisco P.R.G. Hinkkanen A.E. Kohlhaw G.B. Nucleic Acids Res. 1987; 15: 5261-5273Crossref PubMed Scopus (51) Google Scholar). Leu3p binds to target DNA irrespective of the presence or absence of α-IPM (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar). In the absence of α-IPM, Leu3p represses gene expression below the basal level seen in leu3 null cells (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar, 12Sze J.-Y. Woontner M. Jaehning J.A. Kohlhaw G.B. Science. 1992; 258: 1143-1145Crossref PubMed Scopus (78) Google Scholar). In the presence of α-IPM, it causes strong activation. The zinc cluster part of Leu3p's DNA binding domain is located between amino acids 37 and 67 of the 886-residue protein (11Zhou K. Brisco P.R.G. Hinkkanen A.E. Kohlhaw G.B. Nucleic Acids Res. 1987; 15: 5261-5273Crossref PubMed Scopus (51) Google Scholar, 13Bai Y. Kohlhaw G.B. Nucleic Acids Res. 1991; 19: 5991-5997Crossref PubMed Scopus (19) Google Scholar, 14Sze J.-Y. Remboutsika E. Kohlhaw G.B. Mol. Cell. Biol. 1993; 13: 5702-5709Crossref PubMed Scopus (26) Google Scholar), and a putative dimerization domain is found between amino acids 85 and 99 (14Sze J.-Y. Remboutsika E. Kohlhaw G.B. Mol. Cell. Biol. 1993; 13: 5702-5709Crossref PubMed Scopus (26) Google Scholar). A region responsible for transcriptional activation has been identified within the C-terminal 30 amino acids of Leu3p (14Sze J.-Y. Remboutsika E. Kohlhaw G.B. Mol. Cell. Biol. 1993; 13: 5702-5709Crossref PubMed Scopus (26) Google Scholar, 15Zhou K. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 17409-17412Abstract Full Text PDF PubMed Google Scholar). Truncated Leu3p molecules lacking the AD still bind to DNA. They are totally devoid of activation potential and act as repressors (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar, 15Zhou K. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 17409-17412Abstract Full Text PDF PubMed Google Scholar, 16Zhou K. Bai Y. Kohlhaw G.B. Nucleic Acids Res. 1990; 18: 291-298Crossref PubMed Scopus (30) Google Scholar). Deleting as many as 600 amino acids from the middle of Leu3p leaves DNA binding and transcriptional activation functions intact but eliminates modulation (16Zhou K. Bai Y. Kohlhaw G.B. Nucleic Acids Res. 1990; 18: 291-298Crossref PubMed Scopus (30) Google Scholar, 17Friden P. Reynolds C. Schimmel P. Mol. Cell. Biol. 1989; 9: 4056-4060Crossref PubMed Scopus (35) Google Scholar). Such molecules have a constitutively high activation potential that usually exceeds that of modulated forms of Leu3p. The implication of these results is that Leu3p's middle region contains information that is essential for modulation. In dealing with the question of modulation, it may be helpful to break the modulation process down into discrete steps, e.g. the binding of the modulator α-IPM, conformational changes caused by the binding of α-IPM, and the eventual exposure of the AD that allows it to interact with components of the transcription apparatus. Our current view is that the unmasking of the AD as well as its masking in the absence of α-IPM occur without the participation of auxiliary proteins. The strongest support for this notion comes from recent observations made when full-length yeast Leu3p was expressed in mammalian cells (18Guo H. Kohlhaw G.B. FEBS Lett. 1996; 390: 191-195Crossref PubMed Scopus (17) Google Scholar). It was found that the behavior of Leu3p in mouse cells was almost indistinguishable from that seen in its native environment; Leu3p was stable but inactive when α-IPM was absent and caused an apparent severalfold repression of reporter gene expression. When α-IPM was present in the cell culture medium, Leu3p was converted into a strong activator. Since mammalian cells do not synthesize branched chain amino acids, it appeared highly unlikely that they would elaborate Leu3p-specific factors for masking, unmasking, or α-IPM interaction. In this study, we have used biochemical and molecular genetic approaches to advance our understanding of the modulation process. We show that modulation is exquisitely sensitive to mutational changes in the AD of Leu3p. Using wild type Leu3p and a mutant form with drastically intensified masking, we show that the AD and the remainder of Leu3p interact and that this interaction depends on α-IPM. Isolation of intragenic suppressors of the slow growth phenotype of the masking mutant shows the usefulness of this approach for identifying regions or individual residues of Leu3p that are involved in masking. The Saccharomyces cerevisiae strains used were DBY746 (MATα leu2-3leu2-112 trp1-289 ura3-52his3-Δ1; YGSC), DK1 (MATα leu3-Δ::LEU2 leu2-3 leu2-112trp1-289 ura3-52 his3Δ1; see below), and XK157-3C (MATα leu3-Δ2::HIS3 trp1-289 ura3-52 his3-Δ1; (16Zhou K. Bai Y. Kohlhaw G.B. Nucleic Acids Res. 1990; 18: 291-298Crossref PubMed Scopus (30) Google Scholar)). The latter two contain total leu3 deletions. DK1 was constructed as follows. Yeast shuttle vector pRS305 (19Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) was digested withScaI and BsaI. A fragment containing theLEU2 gene was recovered and inserted into plasmid pGB12 (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar) that had been cut with BamHI and rendered blunt-ended with T4 DNA polymerase. The resulting plasmid, pDW1, contained one copy of the LEU2 gene flanked by 5′- and 3′-noncoding sequences ofLEU3. The orientation of LEU2 was the same as the original orientation of LEU3 at this locus. Plasmid pDW1 was digested to completion with SphI and SacI. Among the fragments generated was a 4.3-kbp fragment containing theLEU2 gene and LEU3-flanking sequences. The digestion mixture was used for integrative transformation of strain DBY746, selecting for Leu±. (Since the desired transformants carried an intact LEU2 gene, but noLEU3 gene, the phenotype was expected to change from Leu− (DBY746, no growth in the absence of added leucine) to Leu± (slow growth, about 40% that of wild type cells, Ref. 9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar).) Transformants that also had the His−, Ura−, and Trp− phenotype were collected and designated DK1. Their genotype was further confirmed by PCR using purified yeast chromosomal DNA and primers complementary to theLEU2 coding region and regions of the LEU3 locus flanking the LEU2 gene. Unless stated otherwise, yeast cells were grown on SD medium (20Fink G.R. Methods Enzymol. 1970; 17: 59-78Crossref Scopus (134) Google Scholar) supplemented with the required nutrients. A supplement of 2 mm leucine plus 1 mm each of valine and isoleucine was routinely used to generate low intracellular α-IPM levels. (The presence of valine was found not to be obligatory, and valine was therefore omitted in some experiments.) High α-IPM levels were generated by supplementing the medium with 0.2 mm leucine. Cells were grown at 30 °C and harvested at an A 600 of about 1. Escherichia coli strains used for DNA manipulations were TG1 (K12 [Δlac-pro] supE hsdΔS/F′traΔ36proA + B + lacI q lacZM15), DH5α (Life Technologies, Inc.), and XL-2blue (Stratagene). Strain CJ236 (dut1 ung1 thi-1 relA1/pCJ105[CMr]) was used for isolation of uracil-containing single-stranded DNA. E. colicells were grown at 37 °C in L broth or 2 × YT media with the addition of 100 μg/ml penicillin where needed. The majority of the mutants was generated by degenerate oligonucleotide incorporation. This approach required the construction of an expression plasmid containing a LEU3 gene with engineered restriction sites that would allow precise cassette exchange of the AD. To achieve this, newNgoMI and PmeI/SmaI sites were created by site-directed mutagenesis. E. coli CJ236 cells were transformed with plasmid pRS316-LEU3 which was constructed by cloning aLEU3-containing fragment into pRS316 (19Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). (The fragment extended from −561 to about +4700 relative to the A at the beginning of the open reading frame of LEU3 (11Zhou K. Brisco P.R.G. Hinkkanen A.E. Kohlhaw G.B. Nucleic Acids Res. 1987; 15: 5261-5273Crossref PubMed Scopus (51) Google Scholar).) Transformed cells were grown to mid-log phase and superinfected with helper phage M13K07 in the presence of uracil. Phage particles were collected and single-stranded DNA isolated according to a protocol from Bio-Rad. Equimolar amounts of two phosphorylated primers (5′ CATCATGGCCGGCTGGGATAAC-3′ for the 5′ side of the AD and 5′-CCCAAGGTTTAAACCCGGGTTCTTTTTTTGCG-3′ for the 3′ side of the AD (NgoI and PmeI/SmaI sites underlined)) were then used to mutagenize pRS316-LEU3. Note that changes made to generate the new restriction sites did not alter the amino acid sequence of Leu3p. Potential mutants were screened for the presence of an additional SmaI site, and positives were sequenced using a double-stranded DNA cycle sequencing kit from Life Technologies, Inc. A 1-kbp fragment of DNA containing newNgoMI and PmeI sites was amplified by PCR using appropriate primers. A BlnI-SmaI piece was excised from the fragment and cloned intoBlnI/SmaI-digested pPC62H/86T-LEU3 from which two existing NgoM1 sites had been removed, resulting in plasmid pYHA. It contains unique NgoMI and PmeI sites that define the AD of the Leu-3 protein. Its LEU3 gene is flanked by ADC1 promoter and terminator sequences, respectively. Plasmid pPC62H/86T-LEU3 had been constructed from centromere-containing plasmid pPC62H/86T (a gift from E. Taparowsky, Purdue University) and pT7-LEU3 (a gift from J.-Y. Sze of this laboratory). pT7-LEU3 was digested with PstI andSmaI, and the 3.1-kbp, LEU3-containing fragment was inserted into pPC62H/86T that had also been cut withPstI and SmaI. To mutagenize the AD of Leu3p, a 93-base pair oligonucleotide, 5′-ATGGCCGGCtgggataactgggaatctgatatggtttggagggatgttgatattttaatgaatgaatttgcgttcaatcccaaGGTTTAAACC-3′ (NgoMI and PmeI sites underlined) was synthesized (Integrated DNA Technologies, Coralville, IA) such that the misincorporation rate at the positions shown in lowercase letters was 4.5% (i.e. the proportion of each of the three non-native nucleotides was 1.5%) or, in a separate experiment, 2.7% (the proportion of each of the three non-native nucleotides was 0.9%). The lowercase letters correspond to amino acid positions 861–885 of Leu3p (11Zhou K. Brisco P.R.G. Hinkkanen A.E. Kohlhaw G.B. Nucleic Acids Res. 1987; 15: 5261-5273Crossref PubMed Scopus (51) Google Scholar). The oligonucleotide mixture was incubated, heated to 85 °C, then cooled to 4 °C over a 1-h period. Nucleotides and Klenow enzyme were added to the annealed mixture which was then incubated on ice for 5 min, at 23 °C for 5 min, and at 37 °C for 20–30 min. The extended and now double-stranded DNA was digested with NgoMI and PmeI. The final product was a mixture of monomeric DNA fragments. These were ligated into plasmid pYHA that had been digested with NgoMI and PmeI. The molar ratio of fragment to plasmid was approximately 3. Aliquots of 10 ng of ligation mixture DNA were used to transform E. coli DH5α cells. DNA was purified from 4 ml of overnight cultures using a QIAprep spin column. Sequencing was done by the double-stranded DNA cycle sequencing procedure (Life Technologies, Inc.) using a [33P]ATP-end-labeled primer (5′-CCCGTTACAACTACAATC-3′). pYHA plasmids carrying mutations in theNgoMI-PmeI region of LEU3 were used to transform yeast strain XK157-3C/pYB1 (leu3 null; pYB1 contains a LEU2′-lacZ reporter gene (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar)). Yeast cells were transformed either with the help of a transformation kit (Zymo Research, Orange, CA) or by the lithium acetate procedure (21Ito H. Fukaka Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Transformants were plated on SD medium (20Fink G.R. Methods Enzymol. 1970; 17: 59-78Crossref Scopus (134) Google Scholar). Single colonies were suspended in 10 ml of SD medium supplemented with 2 mmleucine plus 1 mm isoleucine and grown at 30 °C for 24–30 h. Aliquots of the subcultures were then inoculated into 10 ml of SD medium supplemented with either 0.2 mm leucine (for high intracellular concentrations of α-IPM) or 2 mmleucine plus 1 mm isoleucine (for low α-IPM concentrations). To determine reporter gene activity, harvested cells were resuspended in 0.1 m sodium phosphate buffer, pH 7.0, containing 10 mm KCl, 1 mm MgSO4, and 50 mm β-mercaptoethanol. Aliquots of the suspension (made up to a total volume of 1 ml) were mixed with 20 μl of a 0.1% solution of sodium dodecyl sulfate and 50 μl of chloroform and vortexed vigorously for 15 s. The β-galactosidase activity was then measured following the procedure of Miller (22Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar). Several mutations in the AD of Leu3p were generated by site-directed mutagenesis, as described (15Zhou K. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 17409-17412Abstract Full Text PDF PubMed Google Scholar, 23Zhou, K. (1991) A Molecular Switch: Structure-Function Study of Yeast Regulatory Protein Leu3. Ph.D. thesis, Purdue University.Google Scholar). The two sets of data,i.e. those obtained by the degenerate oligonucleotide method and those obtained by site-directed mutagenesis, were normalized with respect to the wild type controls. Following the procedure of Kunkel et al. (24Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar), uracil-containing single-stranded DNA from pRS316-LEU3 was mutagenized using an oligonucleotide (5′-GTTTGGAGGAACGTTAATATTTTAATG-3′) that contained AAC and AAT triplets in place of the native GATs. Plasmid DNA isolated from several E. coli colonies was then used to transform aleu3 null strain (XK157-3C). Colonies growing slowly on leucine− plates were identified, and the original plasmid preparations were sequenced. There was excellent correlation between slow growth and the D872N/D874N double mutation. Plasmids of this type were designated pRS316-LEU3dd.To transfer the LEU3dd DNA to a plasmid with a different marker and to place the gene behind theADC1 promoter, a cassette exchange was performed between pRS316-LEU3dd and pPC62H/86T-LEU3, as follows: pRS316-LEU3dd was used as template for a PCR reaction to synthesize a DNA fragment extending from the SalI site to the end of the LEU3 gene. The PCR primers were 5′-CCAACAGAAGACATACGGA-3′ (for the 5′ end of the fragment) and 5′-GTAGCACCGCGGTCATTACATA AC-3′ (for the 3′ end of the fragment; KspI restriction site underlined). The PCR product was digested with SalI and KspI and then inserted into pPC62H/86T-LEU3 cut with the same enzymes. The resulting plasmid was designated pPC62H/86T-LEU3dd. A derivative encoding a Leu3-dd protein from which residues 174–773 were deleted was created by digesting pPC62H/86T-LEU3dd with SalI andAvrII, followed by Klenow enzyme treatment and re-ligation. It was designated pPC62H/86T-LEU3ddΔ12. The DNA binding part of the two-hybrid system was designed to contain the extended DNA binding region of Leu3p (DB, residues 1–173) and the adjacent “middle region” (MR, residues 174–773). It was expressed behind theADC1 promoter. An appropriate centromere-containing plasmid was constructed by digesting pPC62H/86T-LEU3 (see above) withAvrII, filling in the overhangs with T4 DNA polymerase, and re-closing the plasmid with T4 DNA ligase. This created an in-frame stop codon at amino acid position 775 of Leu3p and replaced the arginine at position 774 with a serine. The resulting plasmid was designated pPC62H/86T-DB-MR. The activation domain constructs for use in the two-hybrid system were also expressed behind the ADC1 promoter. Plasmid pNLVP16 (a gift from E. Taparowsky, Purdue University) served as starting material for pVP-LEU3-WT-AD. A pair of primers was used to clone, by PCR, a fragment encoding the AD of VP16. The primers 5′-CTGAGCTATTCCTGCAGTAGTGAAGAG-3′ (5′ end primer) and 5′TCGACGGATC GACCTAGGACCCGGGGAA-3′ (3′ end primer) were designed to contain a PstI site and an AvrII site, respectively (underlined). The PCR product was digested withPstI and AvrII and then ligated into plasmid pPC62H/86T-LEU3 that had also been cut with PstI andAvrII, thus fusing the VP16 AD sequence to the N terminus of and in-frame with the extended Leu3p AD sequence (residues 774–886). This yielded pPC62H/86T-VP-LEU3-WT-AD. To transfer the VP-LEU3-WT-AD sequence to plasmid pRS423 (multicopy, different marker; ref. 25Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene ( Amst .). 1992; 110: 119-122Crossref PubMed Scopus (1438) Google Scholar), pPC62H/86T-VP-LEU3-WT-AD was digested with ApaI andPvuII. A 3-kbp ApaI-PvuII fragment containing the VP-LEU3-WT-AD sequence was ligated into pRS423 that had been cut with ApaI and SmaI. The resulting plasmid was designated pVP-LEU3-WT-AD. Plasmid pVP-LEU3-dd-AD was constructed in the same way except that pPC62H/86T-LEU3-dd (see above) was used instead of pPC62H/86T-LEU3. To construct pVP, a plasmid coding for the VP16 AD only, the above PCR product was first digested withAvrII, filled in with T4 DNA polymerase, then digested withPstI. The digested PCR product was inserted into plasmid pPC62H/86T-LEU3 that had been cut with SpeI, filled in, and then cut with PstI. The resulting plasmid, pPC62H/86T-VP, contained an in-frame stop codon behind the VP16 AD sequence. Next, the VP sequence was transferred to pRS423 in the way described above, yielding pVP. The DNA sequence of all junction regions and of the entire PCR-synthesized region of the VP16 AD was confirmed using the Sequenase™ version 2.0 sequencing kit from Amersham Corp. Transformation of yeast cells was performed by the lithium acetate method (21Ito H. Fukaka Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). The recipient strain DK1 was transformed first with the reporter plasmid pYB1 (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar) and then with pPC62H/86T-DB-MR. The resulting doubly-transformed strain was then further transformed with either pRS423 (control), or pVP, or pVP-LEU3-WT-AD, or pVP-LEU3-dd-AD. The transformants were purified and single colonies from different isolates were used to inoculate 2 ml of SD medium supplemented with 1 mm each of leucine, valine, and isoleucine. After the pre-cultures had grown to saturation, cells that originated from the same colony were used to inoculate 10 ml of SD medium supplemented with either 0.2 mm leucine (if high intracellular concentrations of α-IPM were desired) or 4 mm leucine and 2 mm each of valine and isoleucine (if low α-IPM concentrations were desired). Preparation of cell-free extracts and determination of β-galactosidase activity were done as described above (see “Mutagenesis of the AD of Leu3p”). To facilitate the identification of mutants (suppressors of the Leu3-dd phenotype), the MR (encompassing residues 172–772) was divided into three subregions defined by naturally existing restriction sites. The first subregion (SubRI) extended from the SalI to theSpeI site (corresponding to residues 172–469); the second subregion (SubRII) from the SpeI to the NdeI site (residues 470–607); the third subregion (SubRIII) from theNdeI to the AvrII site (residues 608–772). The subregions were subjected to mutagenic PCR (26Cadwell R.C. Joyce G.F. PCR Methods & Applications. 1994; 3: S136-S140Crossref PubMed Scopus (318) Google Scholar) separately. The pairs of primers used for SubRI, SubRII, and SubRIII, respectively, were 5′-CCAACAGAAGACATACGGA-3′ plus 5′-TTTCCAGCACTTTGGGAGG-3′, 5′-AAGTCAATTGGAGATTAGTC-3′ plus 5′-TACCTCCACCTTCCTTTTG-3′, and 5′-GACGTTTAATGCCTCAGTT-3′ plus 5′-GTGTCCTTGATGTCTGTAG-3′. The PCR products (pools of mutated DNA fragments) were digested with the appropriate restriction enzymes and inserted into pPC62H/86T-LEU3dd that had been cut with either SalI and SpeI (SubRI), or SpeI and NdeI (SubRII), orNdeI and AvrII (SubRIII). Thus, any one Leu3p molecule contained only one mutated subregion at most. XK157-3C/pYB1 cells (leu3 null with the LEU2′-′lacZreporter) were transformed with ligation solutions containing either SubRI, SubRII, or SubRIII mutants. The transformed cells were plated on SD medium (20Fink G.R. Methods Enzymol. 1970; 17: 59-78Crossref Scopus (134) Google Scholar) and selected for significantly increased growth rates. Cell-free extracts were prepared and β-galactosidase activities were measured as described above (“Mutagenesis of the AD of Leu3p”). Mutants with elevated β-galactosidase activities were isolated, and the appropriate subregion was subjected to DNA sequence analysis. To determine the phenotype of the MR mutants in the context of a Leu3p molecule with a wild type AD, wild type subregions were replaced with the corresponding mutated subregions by cassette exchange. For example, mutated SubRII's were isolated by cutting mutated pPC62H/86T-LEU3dd molecules with SpeI and NdeI and inserting theSpeI-NdeI fragments into pPC62H/86T-LEU3 that had been cut with the same enzymes. The resulting plasmids contained a mutated SubRII in an otherwise wild type LEU3 gene. Whole-cell extracts used in electrophoretic mobility shift assays and Western blots were prepared using the glass bead method (27Dunn, B., and Wobbe, C. R. (1993) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) Vol. 2, Unit 13.13, Wiley Interscience, New York.Google Scholar). Specifically, cells from 10 ml cultures were harvested at anA 600 of about 1, washed once, resuspended in 300 μl of lysis buffer (0.2 m Tris-HCl, pH 8.0, containing 0.4 m [NH4]2SO4, 5 mm MgCl2, 50 μmZnSO4, 1 mm EDTA, 20% (v/v) glycerol, 0.1% (w/v) Nonidet P-40, 3 mm dithiothreitol, 2 mmbenzamidine, 2 mm phenylmethanesulfonyl fluoride, and 2 μm pepstatin), and transferred to a 1.5-ml Eppendorf tube. About 150 μl of treated glass beads were added to the suspension, and the tube was kept on ice. It was vortexed for 30 s and then left on ice for at least 1 min. The procedure was repeated six times. The mixture was then centrifuged for 20 min at 4 °C (Eppendorf table top centrifuge, 14,000 rpm). The supernatant solution was stored at −80 °C. Protein concentration was determined with the Coomassie Plus Protein Assay Reagent (Pierce). For electrophoretic mobility shift assays, whole-cell extract was incubated for 15 min at 30 °C with 25 mm HEPES-NaOH buffer, pH 7.9, containing 80 mm KCl, 5 mmMgCl2, 1 mm EDTA, 4 mmdithiothreitol, 5% (v/v) glycerol, 1 μg of poly(dI-dC)·poly(dI-dC), 40 μg of bovine serum albumin, 1.4 ng of32P-5′ end-labeled UASLEU-30-mer DNA (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar), and 280 ng of non-labeled, non-binding UASLEU-24-mer DNA (9Brisco P.R.G. Kohlhaw G.B. J. Biol. Chem. 1990; 265: 11667-11675Abstract Full Text PDF PubMed Google Scholar) in a total volume of 40 μl. The solution was then applied to a pre-electrophoresed 4% non-denaturing polyacrylamide gel. Electrophoresis was performed for 2.5 h at 30 mA in buffer consisting of 90 mm Tris base, 90 mmH3BO3, and 2 mm EDTA. Gels were dried and autoradiographed. Western blotting was performed on 15% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. For immunoblotting, the enhanced chemiluminescence kit from Amersh" @default.
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• W2028004003 title "Evidence That Intramolecular Interactions Are Involved in Masking the Activation Domain of Transcriptional Activator Leu3p" @default.
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