Proc. Nail. Acad. Sci. USA Vol. 88, pp. 9443-9447, November 1991 Biochemistry
GCRJ of Saccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif (glycolytic gene expression/coactivators/transcriptonal control)
HENRY V. BAKER Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Box J-266, JHMHC, Gainesville, FL 32610
Communicated by Mark Ptashne, July 26, 1991 (received for review March 21, 1991)
In Saccharomyces cerevisiae, glycolysis enABSTRACT zymes constitute 30-60% of the soluble protein. GCRI gene function is required for high-level glycolytic gene expression. In gcrl mutant strains the levels of most glycolytic enzymes are between 2% and 10% of wild type. Binding sites for the global regulatory protein known as repressor activator protein 1 (RAP1)/general regulatory factor 1 (GRF1)/translation upstream factor (TUF) are found in close proximity to one or more CTTCC sequence motifs in the controlling region of GCRIdependent genes. RAP1/GRF1/TUF-bindlng sites are known to be essential elements of upstream activating sequences that control expression of many glycolytic genes. In this report, I demonstrate that GCRI encodes a DNA binding protein whose ability to bind DNA is dependent on the CTTCC sequence motif. This rinding, in addition to the work of others, suggests that the GCRI gene product and the RAP1/GRFI/TUF gene product act in concert to mediate high-level glycolytic gene expression.
Genes encoding the enzymes of glycolysis are among the most highly expressed genes in Saccharomyces cerevisiae (1, 2). Several common regulatory features of these genes are beginning to emerge. Expression of many genes encoding glycolytic enzymes is severely reduced in strains with either gcrl (3-6) or gcr2 (7) mutations. In gcrl mutant strains, the levels of the affected enzymes are reduced to between 2% and 10% of wild type (4-6), and there is a corresponding reduction in the steady-state mRNA levels that specify affected functions (4, 6, 8). Similar patterns of expression are observed in gcr2 mutant strains; however, the effects of gcr2 mutations on growth are not quite as severe as those of gcrl mutations (7). Both GCRJ and GCR2 have been cloned (6, 7, 9), and GCRI has been sequenced (5, 6). The mechanism(s) by which these genes bring about high-level glycolytic gene expression remains to be elucidated. Several similarities have been observed in the controlling region of genes encoding glycolysis enzymes. Putative or demonstrated binding sites for the regulatory protein known as repressor activator protein 1 (RAP1) (10) or general regulatory factor 1 (GRF1) (11) or translation upstream factor (TUF) (12) (hereafter referred to as RAP1) have been noted in front of a number of genes (8, 13-19), and mutational analysis of several of these sites has demonstrated that RAPi binding is essential for expression (8, 14, 15). In addition to a RAP1-binding site, the upstream activating sequence (UAS) of many of these genes contains a pentamer sequence motif, C1TCC, in close proximity to a RAP1-binding site. Mutational analyses have implicated the CTTCC sequence motif in playing a role in the expression of these genes (13, 20). To date, proteins that interact with the CTTCC sequence The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
motif have not been identified (21). In addition to RAP1binding sites and CTTCC sequence motifs, several glycolytic genes have autonomously replicating sequence-binding factor 1 binding sites in their controlling regions (19, 21). I previously suggested, based on DNA sequence analysis, that GCR1 may encode a DNA binding protein (5). However, DNA band shift analysis using extracts from wild-type and gcrl deletion mutant strains revealed no differences in the pattern of band shifting observed with radiolabeled DNA carrying the UAS of the gene encoding triose-phosphate isomerase (TPI) (8). The inability to detect a differential pattern of shifting with extracts prepared from wild-type and mutant strains may be due to the low-level expression of GCRI itself in the wild-type strain (5). In the current study the GCRI gene product was synthesized in vitro by using a rabbit reticulocyte lysate and used in a series of band shift studies. In this communication, I show that GCRJ encodes a DNA binding protein that binds to DNA carrying the CTTCC DNA sequence motif but not to a related fragment of DNA carrying the sequence CAACC in place of the CTTCC motif.
MATERIALS AND METHODS In Vitro Synthesis of GCR1 and RAP1. Both GCR1 and RAP1 transcripts were expressed in vitro under the control of the SP6 promoter. Plasmid pHB66 was prepared to direct the synthesis of GCR1 RNA. This plasmid carries a fragment of DNA from the GCRI region of the yeast genome, extending from an Afl II restriction endonuclease site (located 136 nucleotides 5' to the translational start of GCRI) to a Bcl I restriction endonuclease site (located 661 nucleotides 3' to the translation termination codon of GCRI) (5), cloned into the Sma I/BamHI site of plasmid pSP19 (BRL). Plasmid pSP56RT7 (15), a generous gift from Alistair Chambers (Oxford University), was used to direct the synthesis of RAP1 RNA. In vitro transcription reactions were carried out in the presence of the cap analog m7G(5')ppp(5')G by using a kit from Promega according to the manufacturer's specifications. The in vitro-derived transcripts were then translated in a rabbit reticulocyte lysate system in the presence of L-[35S]methionine by using a kit obtained from Promega according to the manufacturer's specifications. Translation products were analyzed by SDS/PAGE (22), and the radiolabeled proteins were visualized by autoradiography at -700C. DNA Band Shift Assays. DNA band shift assays based on the procedures of Fried and Crothers (23) and Garner and Revzin (24) were carried out as described (8). The typical reaction was carried out in a 20-.lI volume containing 0.5-1 ng of radiolabeled probe DNA along with poly(dI-dC) at 0.26 Abbreviations: RAP1, repressor activator protein 1; UAS, upstream activating sequence.
9443
9444
=.'- -*.!,:.'b"
Biochemistry: Baker
pug/lt
Proc. Natl. Acad. Sci. USA 88 (1991)
as a nonspecific competitor and from 1 to 10 ,ul of a rabbit reticulocyte lysate. Oligonucleotides used as radiolabeled probes in this study were initially synthesized on an Applied Biosystems 380B DNA synthesizer and then cloned into plasmid DNA. The oligonucleotides were then purified from the plasmid DNA and radiolabeled for use in the band shift assays as described. In experiments in which doublestranded oligonucleotides were used as a specific competitor, complementary oligonucleotides were annealed together; hybridization of the oligonucleotides was monitored spectrophotometrically. In the case of band shift experiments that utilized either preimmune or anti-GCR1 serum, the rabbit reticulocyte lysates were pretreated with serum (1 ul of serum per 10 A.l of rabbit reticulocyte lysate) at 300C for 30 min prior to the addition of the lysate to the band shift reaction mixture. Anti-GCR1 Serum. Antiserum against GCR1 was generated through the use of a GCR1-/9-galactosidase fusion protein. The fusion carried amino acids 14-667 of GCR1 fused to the C terminus of (3-galactosidase. The fusion protein was purified, by virtue of its (3-galactosidase moiety, on a (3-galactosidase affinity column as described by Ullman (25). The fusion protein was then used to immunize rabbits. A complete description of the preparation and characterization of the anti-GCR1 serum will appear elsewhere.
RESULTS Previous studies, using crude extracts of yeast, identified a RAP1-binding site in the TPI controlling region, which is required for expression of TPI (8). Identical band shift patterns are observed with extracts prepared from wild-type and gcrl deletion mutant strains in experiments with DNA carrying the UAS element from TPI. To characterize the TPI controlling region further, RAP1 and GCR1 were synthesized
in vitro and used in a series of band shift experiments. Fig. 1 shows the translation products obtained from a rabbit reticulocyte lysate when synthetic mRNA specifying RAP1 or GCR1 was used. In each case, the predominant band had an apparent molecular mass that corresponds to the molecular mass expected for the proteins. GCRI Encodes a DNA Binding Protein. Fig. 2 shows the results of DNA band shift assays using in vitro-synthesized RAP] and GCRI gene products. Both the synthetic RAP1 and GCR1 proteins gave rise to shifted bands when they were mixed with radiolabeled DNA containing sequence from the TPI controlling region [from position -327 to -377 (26)] known to encode a RAP1-binding site (ACACCCCT'TTTlCT) (8). Two shifted bands were routinely observed with the GCR1 rabbit reticulocyte lysate; however, occasionally a third band was observed with GCR1 rabbit reticulocyte lysates. The appearance of the third band may have been due to degradation of GCR1 in the rabbit reticulocyte lysate. No '7!7
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GCRJ of Saccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif (glycolytic gene expression/coactivators/transcriptonal control)
HENRY V. BAKER Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Box J-266, JHMHC, Gainesville, FL 32610
Communicated by Mark Ptashne, July 26, 1991 (received for review March 21, 1991)
In Saccharomyces cerevisiae, glycolysis enABSTRACT zymes constitute 30-60% of the soluble protein. GCRI gene function is required for high-level glycolytic gene expression. In gcrl mutant strains the levels of most glycolytic enzymes are between 2% and 10% of wild type. Binding sites for the global regulatory protein known as repressor activator protein 1 (RAP1)/general regulatory factor 1 (GRF1)/translation upstream factor (TUF) are found in close proximity to one or more CTTCC sequence motifs in the controlling region of GCRIdependent genes. RAP1/GRF1/TUF-bindlng sites are known to be essential elements of upstream activating sequences that control expression of many glycolytic genes. In this report, I demonstrate that GCRI encodes a DNA binding protein whose ability to bind DNA is dependent on the CTTCC sequence motif. This rinding, in addition to the work of others, suggests that the GCRI gene product and the RAP1/GRFI/TUF gene product act in concert to mediate high-level glycolytic gene expression.
Genes encoding the enzymes of glycolysis are among the most highly expressed genes in Saccharomyces cerevisiae (1, 2). Several common regulatory features of these genes are beginning to emerge. Expression of many genes encoding glycolytic enzymes is severely reduced in strains with either gcrl (3-6) or gcr2 (7) mutations. In gcrl mutant strains, the levels of the affected enzymes are reduced to between 2% and 10% of wild type (4-6), and there is a corresponding reduction in the steady-state mRNA levels that specify affected functions (4, 6, 8). Similar patterns of expression are observed in gcr2 mutant strains; however, the effects of gcr2 mutations on growth are not quite as severe as those of gcrl mutations (7). Both GCRJ and GCR2 have been cloned (6, 7, 9), and GCRI has been sequenced (5, 6). The mechanism(s) by which these genes bring about high-level glycolytic gene expression remains to be elucidated. Several similarities have been observed in the controlling region of genes encoding glycolysis enzymes. Putative or demonstrated binding sites for the regulatory protein known as repressor activator protein 1 (RAP1) (10) or general regulatory factor 1 (GRF1) (11) or translation upstream factor (TUF) (12) (hereafter referred to as RAP1) have been noted in front of a number of genes (8, 13-19), and mutational analysis of several of these sites has demonstrated that RAPi binding is essential for expression (8, 14, 15). In addition to a RAP1-binding site, the upstream activating sequence (UAS) of many of these genes contains a pentamer sequence motif, C1TCC, in close proximity to a RAP1-binding site. Mutational analyses have implicated the CTTCC sequence motif in playing a role in the expression of these genes (13, 20). To date, proteins that interact with the CTTCC sequence The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
motif have not been identified (21). In addition to RAP1binding sites and CTTCC sequence motifs, several glycolytic genes have autonomously replicating sequence-binding factor 1 binding sites in their controlling regions (19, 21). I previously suggested, based on DNA sequence analysis, that GCR1 may encode a DNA binding protein (5). However, DNA band shift analysis using extracts from wild-type and gcrl deletion mutant strains revealed no differences in the pattern of band shifting observed with radiolabeled DNA carrying the UAS of the gene encoding triose-phosphate isomerase (TPI) (8). The inability to detect a differential pattern of shifting with extracts prepared from wild-type and mutant strains may be due to the low-level expression of GCRI itself in the wild-type strain (5). In the current study the GCRI gene product was synthesized in vitro by using a rabbit reticulocyte lysate and used in a series of band shift studies. In this communication, I show that GCRJ encodes a DNA binding protein that binds to DNA carrying the CTTCC DNA sequence motif but not to a related fragment of DNA carrying the sequence CAACC in place of the CTTCC motif.
MATERIALS AND METHODS In Vitro Synthesis of GCR1 and RAP1. Both GCR1 and RAP1 transcripts were expressed in vitro under the control of the SP6 promoter. Plasmid pHB66 was prepared to direct the synthesis of GCR1 RNA. This plasmid carries a fragment of DNA from the GCRI region of the yeast genome, extending from an Afl II restriction endonuclease site (located 136 nucleotides 5' to the translational start of GCRI) to a Bcl I restriction endonuclease site (located 661 nucleotides 3' to the translation termination codon of GCRI) (5), cloned into the Sma I/BamHI site of plasmid pSP19 (BRL). Plasmid pSP56RT7 (15), a generous gift from Alistair Chambers (Oxford University), was used to direct the synthesis of RAP1 RNA. In vitro transcription reactions were carried out in the presence of the cap analog m7G(5')ppp(5')G by using a kit from Promega according to the manufacturer's specifications. The in vitro-derived transcripts were then translated in a rabbit reticulocyte lysate system in the presence of L-[35S]methionine by using a kit obtained from Promega according to the manufacturer's specifications. Translation products were analyzed by SDS/PAGE (22), and the radiolabeled proteins were visualized by autoradiography at -700C. DNA Band Shift Assays. DNA band shift assays based on the procedures of Fried and Crothers (23) and Garner and Revzin (24) were carried out as described (8). The typical reaction was carried out in a 20-.lI volume containing 0.5-1 ng of radiolabeled probe DNA along with poly(dI-dC) at 0.26 Abbreviations: RAP1, repressor activator protein 1; UAS, upstream activating sequence.
9443
9444
=.'- -*.!,:.'b"
Biochemistry: Baker
pug/lt
Proc. Natl. Acad. Sci. USA 88 (1991)
as a nonspecific competitor and from 1 to 10 ,ul of a rabbit reticulocyte lysate. Oligonucleotides used as radiolabeled probes in this study were initially synthesized on an Applied Biosystems 380B DNA synthesizer and then cloned into plasmid DNA. The oligonucleotides were then purified from the plasmid DNA and radiolabeled for use in the band shift assays as described. In experiments in which doublestranded oligonucleotides were used as a specific competitor, complementary oligonucleotides were annealed together; hybridization of the oligonucleotides was monitored spectrophotometrically. In the case of band shift experiments that utilized either preimmune or anti-GCR1 serum, the rabbit reticulocyte lysates were pretreated with serum (1 ul of serum per 10 A.l of rabbit reticulocyte lysate) at 300C for 30 min prior to the addition of the lysate to the band shift reaction mixture. Anti-GCR1 Serum. Antiserum against GCR1 was generated through the use of a GCR1-/9-galactosidase fusion protein. The fusion carried amino acids 14-667 of GCR1 fused to the C terminus of (3-galactosidase. The fusion protein was purified, by virtue of its (3-galactosidase moiety, on a (3-galactosidase affinity column as described by Ullman (25). The fusion protein was then used to immunize rabbits. A complete description of the preparation and characterization of the anti-GCR1 serum will appear elsewhere.
RESULTS Previous studies, using crude extracts of yeast, identified a RAP1-binding site in the TPI controlling region, which is required for expression of TPI (8). Identical band shift patterns are observed with extracts prepared from wild-type and gcrl deletion mutant strains in experiments with DNA carrying the UAS element from TPI. To characterize the TPI controlling region further, RAP1 and GCR1 were synthesized
in vitro and used in a series of band shift experiments. Fig. 1 shows the translation products obtained from a rabbit reticulocyte lysate when synthetic mRNA specifying RAP1 or GCR1 was used. In each case, the predominant band had an apparent molecular mass that corresponds to the molecular mass expected for the proteins. GCRI Encodes a DNA Binding Protein. Fig. 2 shows the results of DNA band shift assays using in vitro-synthesized RAP] and GCRI gene products. Both the synthetic RAP1 and GCR1 proteins gave rise to shifted bands when they were mixed with radiolabeled DNA containing sequence from the TPI controlling region [from position -327 to -377 (26)] known to encode a RAP1-binding site (ACACCCCT'TTTlCT) (8). Two shifted bands were routinely observed with the GCR1 rabbit reticulocyte lysate; however, occasionally a third band was observed with GCR1 rabbit reticulocyte lysates. The appearance of the third band may have been due to degradation of GCR1 in the rabbit reticulocyte lysate. No '7!7
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