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1
Bhat, P. J., i J. E. Hopper. "Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon". Molecular and Cellular Biology 12, nr 6 (czerwiec 1992): 2701–7. http://dx.doi.org/10.1128/mcb.12.6.2701-2707.1992.
Pełny tekst źródłaStreszczenie:
The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL80 and GAL3 proteins. In the absence of galactose, GAL80 inhibits the function of GAL4, presumably by direct binding to the GAL4 protein. The presence of galactose triggers the relief of the GAL80 block. The key to this relief is the GAL3 protein. How GAL3 and galactose activate GAL4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL80 or the GAL80-GAL4 complex. Here we report that overproduction of the GAL3 protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GAL1 protein (galactokinase) also causes constitutivity, consistent with the observations that GAL1 is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GAL1. On the basis of these data, we propose a new model for galactose-induced activation of the GAL4 protein. This model invokes galactose-activation of the GAL3 and GAL1 proteins which in turn elicit an alteration of the GAL80-GAL4 complex to activate GAL4. This model is consistent with all the known features of the system and has important implications for manipulating GAL4-dependent transcriptional activation in vitro.
2
Bhat, P. J., i J. E. Hopper. "Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon." Molecular and Cellular Biology 12, nr 6 (czerwiec 1992): 2701–7. http://dx.doi.org/10.1128/mcb.12.6.2701.
Pełny tekst źródłaStreszczenie:
The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL80 and GAL3 proteins. In the absence of galactose, GAL80 inhibits the function of GAL4, presumably by direct binding to the GAL4 protein. The presence of galactose triggers the relief of the GAL80 block. The key to this relief is the GAL3 protein. How GAL3 and galactose activate GAL4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL80 or the GAL80-GAL4 complex. Here we report that overproduction of the GAL3 protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GAL1 protein (galactokinase) also causes constitutivity, consistent with the observations that GAL1 is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GAL1. On the basis of these data, we propose a new model for galactose-induced activation of the GAL4 protein. This model invokes galactose-activation of the GAL3 and GAL1 proteins which in turn elicit an alteration of the GAL80-GAL4 complex to activate GAL4. This model is consistent with all the known features of the system and has important implications for manipulating GAL4-dependent transcriptional activation in vitro.
3
Bajwa, W., T. E. Torchia i J. E. Hopper. "Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases". Molecular and Cellular Biology 8, nr 8 (sierpień 1988): 3439–47. http://dx.doi.org/10.1128/mcb.8.8.3439-3447.1988.
Pełny tekst źródłaStreszczenie:
GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GAL1, GAL7, GAL10, and MEL1 genes, the GAL3 gene is severely glucose repressed. Like the MEL1 gene, but in contrast to the GAL1, GAL7, and GAL10 genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGal elements of the other galactose-melibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GAL1-specified galactokinase of Saccharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.
4
Bajwa, W., T. E. Torchia i J. E. Hopper. "Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases." Molecular and Cellular Biology 8, nr 8 (sierpień 1988): 3439–47. http://dx.doi.org/10.1128/mcb.8.8.3439.
Pełny tekst źródłaStreszczenie:
GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GAL1, GAL7, GAL10, and MEL1 genes, the GAL3 gene is severely glucose repressed. Like the MEL1 gene, but in contrast to the GAL1, GAL7, and GAL10 genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGal elements of the other galactose-melibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GAL1-specified galactokinase of Saccharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.
5
Oh, D., i J. E. Hopper. "Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible". Molecular and Cellular Biology 10, nr 4 (kwiecień 1990): 1415–22. http://dx.doi.org/10.1128/mcb.10.4.1415-1422.1990.
Pełny tekst źródłaStreszczenie:
The Saccharomyces cerevisiae GAL5 (PGM2) gene was isolated and shown to encode the major isozyme of phosphoglucomutase. Northern (RNA) blot hybridization revealed that the GAL5 transcript level increased three- to fourfold in response to galactose and was severely repressed in response to glucose. Total cellular phosphoglucomutase activity was likewise responsive to galactose and to glucose, and this responsiveness was found to be due primarily to variation in the activity of the major isozyme of phosphoglucomutase. These results imply that the major and minor isozymes of phosphoglucomutase have distinct roles in yeast cells. The galactose inducibility of GAL5 was found to be under the control of the GAL4, GAL80, and GAL3 genes. In striking contrast to other galactose-inducible genes, the GAL5 gene exhibited an unusually high GAL4-independent basal level of expression. These results have implications for metabolic trafficking.
6
Oh, D., i J. E. Hopper. "Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible." Molecular and Cellular Biology 10, nr 4 (kwiecień 1990): 1415–22. http://dx.doi.org/10.1128/mcb.10.4.1415.
Pełny tekst źródłaStreszczenie:
The Saccharomyces cerevisiae GAL5 (PGM2) gene was isolated and shown to encode the major isozyme of phosphoglucomutase. Northern (RNA) blot hybridization revealed that the GAL5 transcript level increased three- to fourfold in response to galactose and was severely repressed in response to glucose. Total cellular phosphoglucomutase activity was likewise responsive to galactose and to glucose, and this responsiveness was found to be due primarily to variation in the activity of the major isozyme of phosphoglucomutase. These results imply that the major and minor isozymes of phosphoglucomutase have distinct roles in yeast cells. The galactose inducibility of GAL5 was found to be under the control of the GAL4, GAL80, and GAL3 genes. In striking contrast to other galactose-inducible genes, the GAL5 gene exhibited an unusually high GAL4-independent basal level of expression. These results have implications for metabolic trafficking.
7
Uemura, H., i Y. Jigami. "Role of GCR2 in transcriptional activation of yeast glycolytic genes". Molecular and Cellular Biology 12, nr 9 (wrzesień 1992): 3834–42. http://dx.doi.org/10.1128/mcb.12.9.3834-3842.1992.
Pełny tekst źródłaStreszczenie:
The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.
8
Uemura, H., i Y. Jigami. "Role of GCR2 in transcriptional activation of yeast glycolytic genes." Molecular and Cellular Biology 12, nr 9 (wrzesień 1992): 3834–42. http://dx.doi.org/10.1128/mcb.12.9.3834.
Pełny tekst źródłaStreszczenie:
The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.
9
Bhat, P. J., D. Oh i J. E. Hopper. "Analysis of the GAL3 signal transduction pathway activating GAL4 protein-dependent transcription in Saccharomyces cerevisiae." Genetics 125, nr 2 (1.06.1990): 281–91. http://dx.doi.org/10.1093/genetics/125.2.281.
Pełny tekst źródłaStreszczenie:
Abstract The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.
10
Finley, R. L., S. Chen, J. Ma, P. Byrne i R. W. West. "Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes". Molecular and Cellular Biology 10, nr 11 (listopad 1990): 5663–70. http://dx.doi.org/10.1128/mcb.10.11.5663-5670.1990.
Pełny tekst źródłaStreszczenie:
The yeast GAL1 and GAL10 genes are transcribed at a remarkably low basal level when galactose is unavailable and are induced by over 4 orders of magnitude when it becomes available. Approximately six negative control elements (designated GAL operators GALO1 to GALO6) are located adjacent to or overlapping four binding sites for the transcription activator GAL4 in the GAL upstream activating sequence UASG. The negative control elements contribute to the broad range of inducibility of GAL1 and GAL10 by inhibiting two GAL4/galactose-independent activating elements (GAE1 and GAE2) in UASG. In turn, multiple GAL4-binding sites in UASG are necessary for GAL4 to overcome repression by the negative control elements under fully inducing conditions. When glucose in addition to galactose is available (repressing conditions), the ability of GAL4 to activate transcription is diminished as a result of its reduced affinity for DNA and the reduced availability of inducer. Under these conditions, the negative control elements inhibit transcriptional activation from the glucose-attenuated GAL4 sites, thus accounting at least in part for glucose repression acting in cis. A normal part of transcriptional regulation of the GAL1 and GAL10 genes, therefore, appears to involve a balance between the opposing functions of positive and negative control elements.
11
Finley, R. L., i R. W. West. "Differential repression of GAL4 and adjacent transcription activators by operators in the yeast GAL upstream activating sequence". Molecular and Cellular Biology 9, nr 10 (październik 1989): 4282–90. http://dx.doi.org/10.1128/mcb.9.10.4282-4290.1989.
Pełny tekst źródłaStreszczenie:
The upstream activating sequence of the adjacent and divergently transcribed GAL1 and GAL10 genes of Saccharomyces cerevisiae (UASG) contains at least three distinct classes of overlapping transcriptional control sites. The transcription activator GAL4 binds to four related sites in UASG and induces expression of GAL1 and GAL10 when galactose is available. We showed that UASG contains two additional positive control sites, designated GAL4/galactose-independent activating elements (GAEs), which reside at positions adjacent to or overlapping the GAL4-binding sites. When separated from neighboring sequences in UASG, the GAEs activate transcription independently of GAL4 with no requirement for galactose. In the intact GAL1-GAL10 divergent promoter region, their activity is ordinarily repressed by multiple negative control elements, the GAL operators. When galactose is available, GAL4 overcomes the activity of the GAL operators, while the putative GAE-binding proteins stay repressed. Combined, these results imply that distinct activators (GAL4 and GAE proteins) bound at adjacent or overlapping sites in UASG are differentially regulated by putative repressor proteins simultaneously bound at adjacent GAL operators. We surmise that GAE1 and GAE2 may have a physiological function other than regulation of galactose catabolism per se and discuss three hypotheses to account for their presence in UASG.
12
Finley, R. L., S. Chen, J. Ma, P. Byrne i R. W. West. "Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes." Molecular and Cellular Biology 10, nr 11 (listopad 1990): 5663–70. http://dx.doi.org/10.1128/mcb.10.11.5663.
Pełny tekst źródłaStreszczenie:
The yeast GAL1 and GAL10 genes are transcribed at a remarkably low basal level when galactose is unavailable and are induced by over 4 orders of magnitude when it becomes available. Approximately six negative control elements (designated GAL operators GALO1 to GALO6) are located adjacent to or overlapping four binding sites for the transcription activator GAL4 in the GAL upstream activating sequence UASG. The negative control elements contribute to the broad range of inducibility of GAL1 and GAL10 by inhibiting two GAL4/galactose-independent activating elements (GAE1 and GAE2) in UASG. In turn, multiple GAL4-binding sites in UASG are necessary for GAL4 to overcome repression by the negative control elements under fully inducing conditions. When glucose in addition to galactose is available (repressing conditions), the ability of GAL4 to activate transcription is diminished as a result of its reduced affinity for DNA and the reduced availability of inducer. Under these conditions, the negative control elements inhibit transcriptional activation from the glucose-attenuated GAL4 sites, thus accounting at least in part for glucose repression acting in cis. A normal part of transcriptional regulation of the GAL1 and GAL10 genes, therefore, appears to involve a balance between the opposing functions of positive and negative control elements.
13
Finley, R. L., i R. W. West. "Differential repression of GAL4 and adjacent transcription activators by operators in the yeast GAL upstream activating sequence." Molecular and Cellular Biology 9, nr 10 (październik 1989): 4282–90. http://dx.doi.org/10.1128/mcb.9.10.4282.
Pełny tekst źródłaStreszczenie:
The upstream activating sequence of the adjacent and divergently transcribed GAL1 and GAL10 genes of Saccharomyces cerevisiae (UASG) contains at least three distinct classes of overlapping transcriptional control sites. The transcription activator GAL4 binds to four related sites in UASG and induces expression of GAL1 and GAL10 when galactose is available. We showed that UASG contains two additional positive control sites, designated GAL4/galactose-independent activating elements (GAEs), which reside at positions adjacent to or overlapping the GAL4-binding sites. When separated from neighboring sequences in UASG, the GAEs activate transcription independently of GAL4 with no requirement for galactose. In the intact GAL1-GAL10 divergent promoter region, their activity is ordinarily repressed by multiple negative control elements, the GAL operators. When galactose is available, GAL4 overcomes the activity of the GAL operators, while the putative GAE-binding proteins stay repressed. Combined, these results imply that distinct activators (GAL4 and GAE proteins) bound at adjacent or overlapping sites in UASG are differentially regulated by putative repressor proteins simultaneously bound at adjacent GAL operators. We surmise that GAE1 and GAE2 may have a physiological function other than regulation of galactose catabolism per se and discuss three hypotheses to account for their presence in UASG.
14
Mylin, L. M., M. Johnston i J. E. Hopper. "Phosphorylated forms of GAL4 are correlated with ability to activate transcription". Molecular and Cellular Biology 10, nr 9 (wrzesień 1990): 4623–29. http://dx.doi.org/10.1128/mcb.10.9.4623-4629.1990.
Pełny tekst źródłaStreszczenie:
GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.
15
Mylin, L. M., M. Johnston i J. E. Hopper. "Phosphorylated forms of GAL4 are correlated with ability to activate transcription." Molecular and Cellular Biology 10, nr 9 (wrzesień 1990): 4623–29. http://dx.doi.org/10.1128/mcb.10.9.4623.
Pełny tekst źródłaStreszczenie:
GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.
16
Salmeron, J. M., K. K. Leuther i S. A. Johnston. "GAL4 mutations that separate the transcriptional activation and GAL80-interactive functions of the yeast GAL4 protein." Genetics 125, nr 1 (1.05.1990): 21–27. http://dx.doi.org/10.1093/genetics/125.1.21.
Pełny tekst źródłaStreszczenie:
Abstract The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.
17
Bhat, P. J., i J. E. Hopper. "The mechanism of inducer formation in gal3 mutants of the yeast galactose system is independent of normal galactose metabolism and mitochondrial respiratory function." Genetics 128, nr 2 (1.06.1991): 233–39. http://dx.doi.org/10.1093/genetics/128.2.233.
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Abstract Saccharomyces cerevisiae cells defective in GAL3 function exhibit either one of two phenotypes. The gal3 mutation in an otherwise normal cell causes a 2-5-day delay in the galactose triggered induction of GAL/MEL gene transcription. This long term adaptation (LTA) phenotype has been ascribed to inefficient inducer formation. The gal3 mutation causes a noninducible phenotype for GAL/MEL transcription if cells are defective in Leloir pathway function, in glycolysis or in respiratory function. It was recently shown that multiple copies of the intact GAL1 gene partially suppress the LTA phenotype of gal3 cells. Here we report that constitutively expressed GAL1 restored gal3 mutants to the rapidly inducible phenotype characteristic of wild-type cells and conferred rapid inducibility to gal3 gal10, gal3 gal7 or gal3 rho- strains that are normally noninducible. As shown by immunoblot analysis, the GAL1-mediated induction exhibits phosphorylation of the GAL4 protein, suggesting a mechanism similar to GAL3-mediated induction. Altogether our results indicate that the deciding factor in the inducibility of the GAL/MEL genes in gal3 strains is the Gal3p-like activity of Gal1p. Based on the above we conclude that inducer formation does not require normal metabolism of galactose nor does it require mitochondrial respiratory function. These conclusions vitiate previous explanations for gal3 associated long-term adaptation and noninducible phenotypes.
18
Selleck, S. B., i J. E. Majors. "In vivo DNA-binding properties of a yeast transcription activator protein". Molecular and Cellular Biology 7, nr 9 (wrzesień 1987): 3260–67. http://dx.doi.org/10.1128/mcb.7.9.3260-3267.1987.
Pełny tekst źródłaStreszczenie:
UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.
19
Selleck, S. B., i J. E. Majors. "In vivo DNA-binding properties of a yeast transcription activator protein." Molecular and Cellular Biology 7, nr 9 (wrzesień 1987): 3260–67. http://dx.doi.org/10.1128/mcb.7.9.3260.
Pełny tekst źródłaStreszczenie:
UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.
20
Suzuki, Y., Y. Nogi, A. Abe i T. Fukasawa. "GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae". Molecular and Cellular Biology 8, nr 11 (listopad 1988): 4991–99. http://dx.doi.org/10.1128/mcb.8.11.4991-4999.1988.
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Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.
21
Suzuki, Y., Y. Nogi, A. Abe i T. Fukasawa. "GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae." Molecular and Cellular Biology 8, nr 11 (listopad 1988): 4991–99. http://dx.doi.org/10.1128/mcb.8.11.4991.
Pełny tekst źródłaStreszczenie:
Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.
22
Long, R. M., L. M. Mylin i J. E. Hopper. "GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator". Molecular and Cellular Biology 11, nr 4 (kwiecień 1991): 2311–14. http://dx.doi.org/10.1128/mcb.11.4.2311-2314.1991.
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The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.
23
Long, R. M., L. M. Mylin i J. E. Hopper. "GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator." Molecular and Cellular Biology 11, nr 4 (kwiecień 1991): 2311–14. http://dx.doi.org/10.1128/mcb.11.4.2311.
Pełny tekst źródłaStreszczenie:
The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.
24
Li, Qiming, i Stephen Albert Johnston. "Are All DNA Binding and Transcription Regulation by an Activator Physiologically Relevant?" Molecular and Cellular Biology 21, nr 7 (1.04.2001): 2467–74. http://dx.doi.org/10.1128/mcb.21.7.2467-2474.2001.
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ABSTRACT Understanding how a regulatory protein occupies its sites in vivo is central to understanding gene regulation. Using the yeast Gal4 protein as a model for such studies, we have found 239 potential Gal4 binding sites in the yeast genome, 186 of which are in open reading frames (ORFs). This raises the questions of whether these sites are occupied by Gal4 and, if so, to what effect. We have analyzed theSaccharomyces cerevisiae ACC1 gene (encoding acetyl-coenzyme A carboxylase), which has three Gal4 binding sites in its ORF. The plasmid titration assay has demonstrated that Gal4 occupies these sites in the context of an active ACC1gene. We also find that the expression of the ACC1 is reduced fourfold in galactose medium and that this reduction is dependent on the Gal4 binding sites, suggesting that Gal4 bound to the ORF sites affects transcription of ACC1. Interestingly, removal of the Gal4 binding sites has no obvious effect on the growth in galactose under laboratory conditions. In addition, though the sequence of the ACC1 gene is highly conserved among yeast species, these Gal4 binding sites are not present in theKluyveromyces lactis ACC1 gene. We suggest that the occurrence of these sites may not be related to galactose regulation and a manifestation of the “noise” in the occurrence of Gal4 binding sites.
25
Harbury, P. A., i K. Struhl. "Functional distinctions between yeast TATA elements". Molecular and Cellular Biology 9, nr 12 (grudzień 1989): 5298–304. http://dx.doi.org/10.1128/mcb.9.12.5298-5304.1989.
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Although the yeast his3 promoter region contains two functional TATA elements, TR and TC, the GCN4 and GAL4 upstream activator proteins stimulate transcription only through TR. In combination with GAL4, an oligonucleotide containing the sequence TATAAA is fully sufficient for TR function, whereas almost all single-base-pair substitutions of this sequence abolish the ability of this element to activate transcription. Further analysis of these and other mutations of the TR element led to the following conclusions. First, sequences downstream of the TATAAA sequence are important for TR function. Second, a double mutant, TATTTA, can serve as a TR element even though the corresponding single mutation, TATTAA, is unable to do so. Third, three mutations have the novel property of being able to activate transcription in combination with GCN4 but not with GAL4; this finding suggests that activation by GCN4 and by GAL4 may not occur by identical mechanisms. From these observations, we address the question of whether there is a single TATA-binding factor required for the transcription of all genes.
26
Harbury, P. A., i K. Struhl. "Functional distinctions between yeast TATA elements." Molecular and Cellular Biology 9, nr 12 (grudzień 1989): 5298–304. http://dx.doi.org/10.1128/mcb.9.12.5298.
Pełny tekst źródłaStreszczenie:
Although the yeast his3 promoter region contains two functional TATA elements, TR and TC, the GCN4 and GAL4 upstream activator proteins stimulate transcription only through TR. In combination with GAL4, an oligonucleotide containing the sequence TATAAA is fully sufficient for TR function, whereas almost all single-base-pair substitutions of this sequence abolish the ability of this element to activate transcription. Further analysis of these and other mutations of the TR element led to the following conclusions. First, sequences downstream of the TATAAA sequence are important for TR function. Second, a double mutant, TATTTA, can serve as a TR element even though the corresponding single mutation, TATTAA, is unable to do so. Third, three mutations have the novel property of being able to activate transcription in combination with GCN4 but not with GAL4; this finding suggests that activation by GCN4 and by GAL4 may not occur by identical mechanisms. From these observations, we address the question of whether there is a single TATA-binding factor required for the transcription of all genes.
27
Tchórzewski, M., B. Boldyreff i N. Grankowski. "Extraribosomal function of the acidic ribosomal P1-protein YP1alpha from Saccharomyces cerevisiae." Acta Biochimica Polonica 46, nr 4 (31.12.1999): 901–10. http://dx.doi.org/10.18388/abp.1999_4112.
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The yeast acidic ribosomal P-proteins YP1alpha, YP1beta, YP2alpha and YP2beta were studied for a possible transactivation potential beside their ribosomal function. The fusions of P-proteins with the GAL4 DNA-binding domain were assayed toward their transcriptional activity with the aid of reporter genes in yeast. Two of the P-proteins, YP1alpha and YP1beta, exhibited transactivation potential, however, only YP1alpha can be regarded as a potent transactivator. This protein was able to transactivate a reporter gene associated with two distinct promoter systems, GAL1 or CYC1. Additionally, truncated proteins of YP1alpha and YP1beta were analyzed. The N-terminal part of YP1alpha fused to GAL4-BD showed transactivation potential but the C-terminal part did not. Our results suggest a putative extraribosomal function for these ribosomal proteins which consequently may be classified as "moonlighting" proteins.
28
Traven, Ana, Branka Jelicic i Mary Sopta. "Yeast Gal4: a transcriptional paradigm revisited". EMBO reports 7, nr 5 (maj 2006): 496–99. http://dx.doi.org/10.1038/sj.embor.7400679.
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Chasman, D. I., i R. D. Kornberg. "GAL4 protein: purification, association with GAL80 protein, and conserved domain structure". Molecular and Cellular Biology 10, nr 6 (czerwiec 1990): 2916–23. http://dx.doi.org/10.1128/mcb.10.6.2916-2923.1990.
Pełny tekst źródłaStreszczenie:
Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.
30
Chasman, D. I., i R. D. Kornberg. "GAL4 protein: purification, association with GAL80 protein, and conserved domain structure." Molecular and Cellular Biology 10, nr 6 (czerwiec 1990): 2916–23. http://dx.doi.org/10.1128/mcb.10.6.2916.
Pełny tekst źródłaStreszczenie:
Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.
31
Fedor, M. J., i R. D. Kornberg. "Upstream activation sequence-dependent alteration of chromatin structure and transcription activation of the yeast GAL1-GAL10 genes". Molecular and Cellular Biology 9, nr 4 (kwiecień 1989): 1721–32. http://dx.doi.org/10.1128/mcb.9.4.1721-1732.1989.
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Conversion of the positioned nucleosome array characteristic of the repressed GAL1-GAL10 promoter region to the more accessible conformation of the induced state was found to depend on the upstream activation sequence, GAL4 protein, a positive regulator of transcription, and galactose, the inducing agent. The effect of the GAL4 protein-upstream activation sequence complex on the structure of adjacent chromatin required no other promoter sequences. Although sequences protected by histones in the repressed state became more accessible to micrococcal nuclease and (methidiumpropyl-EDTA)iron(II) cleavage following induction of transcription, DNA-protein particles containing these sequences retained the electrophoretic mobility of nucleosomes, indicating that the promoter region can be associated with nucleosomes under conditions of transcription activation.
32
Fedor, M. J., i R. D. Kornberg. "Upstream activation sequence-dependent alteration of chromatin structure and transcription activation of the yeast GAL1-GAL10 genes." Molecular and Cellular Biology 9, nr 4 (kwiecień 1989): 1721–32. http://dx.doi.org/10.1128/mcb.9.4.1721.
Pełny tekst źródłaStreszczenie:
Conversion of the positioned nucleosome array characteristic of the repressed GAL1-GAL10 promoter region to the more accessible conformation of the induced state was found to depend on the upstream activation sequence, GAL4 protein, a positive regulator of transcription, and galactose, the inducing agent. The effect of the GAL4 protein-upstream activation sequence complex on the structure of adjacent chromatin required no other promoter sequences. Although sequences protected by histones in the repressed state became more accessible to micrococcal nuclease and (methidiumpropyl-EDTA)iron(II) cleavage following induction of transcription, DNA-protein particles containing these sequences retained the electrophoretic mobility of nucleosomes, indicating that the promoter region can be associated with nucleosomes under conditions of transcription activation.
33
Griggs, D. W., i M. Johnston. "Promoter elements determining weak expression of the GAL4 regulatory gene of Saccharomyces cerevisiae". Molecular and Cellular Biology 13, nr 8 (sierpień 1993): 4999–5009. http://dx.doi.org/10.1128/mcb.13.8.4999-5009.1993.
Pełny tekst źródłaStreszczenie:
The GAL4 gene of Saccharomyces cerevisiae (encoding the activator of transcription of the GAL genes) is poorly expressed and is repressed during growth on glucose. To determine the basis for its weak expression and to identify DNA sequences recognized by proteins that activate transcription of a gene that itself encodes an activator of transcription, we have analyzed GAL4 promoter structure. We show that the GAL4 promoter is about 90-fold weaker than the strong GAL1 promoter and at least 7-fold weaker than the feeble URA3 promoter and that this low level of GAL4 expression is primarily due to a weak promoter. By deletion mapping, the GAL4 promoter can be divided into three functional regions. Two of these regions contain positive elements; a distal region termed the UASGAL4 (upstream activation sequence) contains redundant elements that increase promoter function, and a central region termed the UESGAL4 (upstream essential sequence) is essential for even basal levels of GAL4 expression. The third element, an upstream repression sequence, mediates glucose repression of GAL4 expression and is located between the UES and the transcriptional start site. The UASGAL4 is unusual because it is not interchangable with UAS elements in other yeast promoters; it does not function as a UAS element when inserted in a CYC1 promoter, and a normally strong UAS functions poorly in place of UASGAL4 in the GAL4 promoter. Similarly, the UES element of GAL4 does not function as a TATA element in a test promoter, and consensus TATA elements do not function in place of UES elements in the GAL4 promoter. These results suggest that GAL4 contains a weak TATA-less promoter and that the proteins regulating expression of this regulatory gene may be novel and context specific.
34
Griggs, D. W., i M. Johnston. "Promoter elements determining weak expression of the GAL4 regulatory gene of Saccharomyces cerevisiae." Molecular and Cellular Biology 13, nr 8 (sierpień 1993): 4999–5009. http://dx.doi.org/10.1128/mcb.13.8.4999.
Pełny tekst źródłaStreszczenie:
The GAL4 gene of Saccharomyces cerevisiae (encoding the activator of transcription of the GAL genes) is poorly expressed and is repressed during growth on glucose. To determine the basis for its weak expression and to identify DNA sequences recognized by proteins that activate transcription of a gene that itself encodes an activator of transcription, we have analyzed GAL4 promoter structure. We show that the GAL4 promoter is about 90-fold weaker than the strong GAL1 promoter and at least 7-fold weaker than the feeble URA3 promoter and that this low level of GAL4 expression is primarily due to a weak promoter. By deletion mapping, the GAL4 promoter can be divided into three functional regions. Two of these regions contain positive elements; a distal region termed the UASGAL4 (upstream activation sequence) contains redundant elements that increase promoter function, and a central region termed the UESGAL4 (upstream essential sequence) is essential for even basal levels of GAL4 expression. The third element, an upstream repression sequence, mediates glucose repression of GAL4 expression and is located between the UES and the transcriptional start site. The UASGAL4 is unusual because it is not interchangable with UAS elements in other yeast promoters; it does not function as a UAS element when inserted in a CYC1 promoter, and a normally strong UAS functions poorly in place of UASGAL4 in the GAL4 promoter. Similarly, the UES element of GAL4 does not function as a TATA element in a test promoter, and consensus TATA elements do not function in place of UES elements in the GAL4 promoter. These results suggest that GAL4 contains a weak TATA-less promoter and that the proteins regulating expression of this regulatory gene may be novel and context specific.
35
Parthun, M. R., i J. A. Jaehning. "A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80". Molecular and Cellular Biology 12, nr 11 (listopad 1992): 4981–87. http://dx.doi.org/10.1128/mcb.12.11.4981-4987.1992.
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The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.
36
Parthun, M. R., i J. A. Jaehning. "A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80." Molecular and Cellular Biology 12, nr 11 (listopad 1992): 4981–87. http://dx.doi.org/10.1128/mcb.12.11.4981.
Pełny tekst źródłaStreszczenie:
The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.
37
Parthun, M. R., D. A. Mangus i J. A. Jaehning. "The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding". Molecular and Cellular Biology 12, nr 12 (grudzień 1992): 5683–89. http://dx.doi.org/10.1128/mcb.12.12.5683-5689.1992.
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A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.
38
Parthun, M. R., D. A. Mangus i J. A. Jaehning. "The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding." Molecular and Cellular Biology 12, nr 12 (grudzień 1992): 5683–89. http://dx.doi.org/10.1128/mcb.12.12.5683.
Pełny tekst źródłaStreszczenie:
A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.
39
Gustafson, Kerstin, i Gabrielle L. Boulianne. "Distinct expression patterns detected within individual tissues by the GAL4 enhancer trap technique". Genome 39, nr 1 (1.02.1996): 174–82. http://dx.doi.org/10.1139/g96-023.
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To identify genes that are expressed in specific cell types or tissues during development, we generated enhancer-trap lines in which the yeast transcriptional activator, GAL4, was mobilized throughout the Drosophila genome. The GAL4 lines are part of a two-part system involving GAL4 and its target, the upstream activating sequence (UAS). Detection of GAL4 expression patterns was achieved by crossing individual GAL4 lines with flies carrying the reporter gene lacZ under the transcriptional control of the UAS followed by histochemical and immunocytochemical staining. Here, we present the results of this screen and the characterization of GAL4 lines that show distinct patterns of gene expression during Drosophila development, including embryogenesis, oogenesis, and imaginai disc development. However, we were unable to identify GAL4 lines that were expressed within the germ line or during early embryogenesis. Furthermore, consistent with previous results, we found that the GAL4 enhancer trap technique had a much lower frequency of transposition than has been reported for lacZ enhancer trap screens. Taken together, these results demonstrate both the strengths and weaknesses of the GAL4 enhancer trap technique for identifying unique patterns of gene expression during development. Key words : GAL4, enhancer trap, Drosophila, P element.
40
Johnston, S. A., M. J. Zavortink, C. Debouck i J. E. Hopper. "Functional domains of the yeast regulatory protein GAL4." Proceedings of the National Academy of Sciences 83, nr 17 (1.09.1986): 6553–57. http://dx.doi.org/10.1073/pnas.83.17.6553.
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Burns, L. G., i C. L. Peterson. "The yeast SWI-SNF complex facilitates binding of a transcriptional activator to nucleosomal sites in vivo." Molecular and Cellular Biology 17, nr 8 (sierpień 1997): 4811–19. http://dx.doi.org/10.1128/mcb.17.8.4811.
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The Saccharomyces cerevisiae SWI-SNF complex is a 2-MDa protein assembly that is required for the function of many transcriptional activators. Here we describe experiments on the role of the SWI-SNF complex in activation of transcription by the yeast activator GAL4. We find that while SWI-SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind to and function at low-affinity, nucleosomal binding sites in vivo. This SWI-SNF dependence can be overcome by (i) replacing the low-affinity sites with higher-affinity, consensus GAL4 binding sequences or (ii) placing the low-affinity sites into a nucleosome-free region. These results define the criteria for the SWI-SNF dependence of gene expression and provide the first in vivo evidence that the SWI-SNF complex can regulate gene expression by modulating the DNA binding of an upstream activator protein.
42
Stafford, Grace A., i Randall H. Morse. "GCN5 Dependence of Chromatin Remodeling and Transcriptional Activation by the GAL4 and VP16 Activation Domains in Budding Yeast". Molecular and Cellular Biology 21, nr 14 (15.07.2001): 4568–78. http://dx.doi.org/10.1128/mcb.21.14.4568-4578.2001.
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ABSTRACT Chromatin-modifying enzymes such as the histone acetyltransferase GCN5 can contribute to transcriptional activation at steps subsequent to the initial binding of transcriptional activators. However, few studies have directly examined dependence of chromatin remodeling in vivo on GCN5 or other acetyltransferases, and none have examined remodeling via nucleosomal activator binding sites. In this study, we have monitored chromatin perturbation via nucleosomal binding sites in the yeast episome TALS by GAL4 derivatives in GCN5+ andgcn5Δ yeast cells. The strong activator GAL4 shows no dependence on GCN5 for remodeling TALS chromatin, whereas GAL4-estrogen receptor-VP16 shows substantial, albeit not complete, GCN5 dependence. Mini-GAL4 derivatives having weakened interactions with TATA-binding protein and TFIIB exhibit a strong dependence on GCN5 for both transcriptional activation and TALS remodeling not seen for native GAL4. These results indicate that GCN5 can contribute to chromatin remodeling at activator binding sites and that dependence on coactivator function for a given activator can vary according to the type and strength of contacts that it makes with other factors. We also found a weaker dependence for chromatin remodeling on SPT7 than on GCN5, indicating that GCN5 can function via pathways independent of the SAGA complex. Finally, we examine dependence on GCN5 and SWI-SNF at two model promoters and find that although these two chromatin-remodeling and/or modification activities may sometimes work together, in other instances they act in complementary fashion.
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McDonnell, D. P., Z. Nawaz i B. W. O'Malley. "In situ distinction between steroid receptor binding and transactivation at a target gene". Molecular and Cellular Biology 11, nr 9 (wrzesień 1991): 4350–55. http://dx.doi.org/10.1128/mcb.11.9.4350-4355.1991.
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We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen. Inhibition occurs thought the disruption of complex cooperative interactions among the GAL4 molecules in this reporter. The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding. Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand. In contrast, this latter receptor form will not activate transcription. Our results are consistent with a two-step process for receptor activation. Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form. Second, it converts receptor to a transcriptionally competent form. With use of this yeast model system, these two steps can be distinguished in situ.
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McDonnell, D. P., Z. Nawaz i B. W. O'Malley. "In situ distinction between steroid receptor binding and transactivation at a target gene." Molecular and Cellular Biology 11, nr 9 (wrzesień 1991): 4350–55. http://dx.doi.org/10.1128/mcb.11.9.4350.
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We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen. Inhibition occurs thought the disruption of complex cooperative interactions among the GAL4 molecules in this reporter. The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding. Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand. In contrast, this latter receptor form will not activate transcription. Our results are consistent with a two-step process for receptor activation. Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form. Second, it converts receptor to a transcriptionally competent form. With use of this yeast model system, these two steps can be distinguished in situ.
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Sil, Alok Kumar, Samina Alam, Ping Xin, Ly Ma, Melissa Morgan, Colleen M. Lebo, Michael P. Woods i James E. Hopper. "The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP". Molecular and Cellular Biology 19, nr 11 (1.11.1999): 7828–40. http://dx.doi.org/10.1128/mcb.19.11.7828.
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ABSTRACT The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation ofGAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.
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Zhang, Daoyong, i Lu Bai. "Interallelic interaction and gene regulation in budding yeast". Proceedings of the National Academy of Sciences 113, nr 16 (4.04.2016): 4428–33. http://dx.doi.org/10.1073/pnas.1601003113.
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In Drosophila, homologous chromosome pairing leads to “transvection,” in which the enhancer of a gene can regulate the allelic transcription in trans. Interallelic interactions were also observed in vegetative diploid budding yeast, but their functional significance is unknown. Here, we show that a GAL1 reporter can interact with its homologous allele and affect its expression. By ectopically inserting two allelic reporters, one driven by wild-type GAL1 promoter (WT GAL1pr) and the other by a mutant promoter with delayed response to galactose induction, we found that the two reporters physically associate, and the WT GAL1pr triggers synchronized firing of the defective promoter and accelerates its activation without affecting its steady-state expression level. This interaction and the transregulatory effect disappear when the same reporters are located at nonallelic sites. We further demonstrated that the activator Gal4 is essential for the interallelic interaction, and the transregulation requires fully activated WT GAL1pr transcription. The mechanism of this phenomenon was further discussed. Taken together, our data revealed the existence of interallelic gene regulation in yeast, which serves as a starting point for understanding long-distance gene regulation in this genetically tractable system.
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Seol, W., M. Chung i D. D. Moore. "Novel receptor interaction and repression domains in the orphan receptor SHP." Molecular and Cellular Biology 17, nr 12 (grudzień 1997): 7126–31. http://dx.doi.org/10.1128/mcb.17.12.7126.
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SHP (short heterodimer partner) is a novel orphan receptor that lacks a conventional DNA binding domain and interacts with other members of the nuclear hormone receptor superfamily. We have characterized the SHP sequences required for interaction with other superfamily members, and have defined an SHP repressor domain. In the mammalian two-hybrid system, a fusion of full-length SHP to the GAL4 DNA binding domain shows 9-cis-retinoic acid-dependent interaction with a VP16-retinoid X receptor alpha (RXR alpha) fusion. By deletion analysis, sequences required for this RXR interaction map to the central portion of SHP (amino acids 92 to 148). The same region is required for interaction with RXR in vitro and in the yeast two-hybrid system, and results from the yeast system suggest that the same SHP sequences are required for interaction with other members of the nuclear hormone receptor superfamily such as thyroid hormone receptor and retinoic acid receptor. In mammalian cells, a GAL4-SHP fusion protein shows about 10-fold-decreased transcriptional activation relative to GAL4 alone, and fusion of SHP to the C terminus of a GAL4-VP16 fusion to generate a triple chimera also results in a strong decrease in transactivation activity. Sequences required for this repressor function were mapped to the C terminus of SHP. This region is distinct from that required for corepressor interaction by other members of the nuclear hormone receptor superfamily, and SHP did not interact with N-CoR in either the yeast or mammalian two-hybrid system. Together, these results identify novel receptor interaction and repressor domains in SHP and suggest two distinct mechanisms for inhibition of receptor signaling pathways by SHP.
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Candau, R., P. A. Moore, L. Wang, N. Barlev, C. Y. Ying, C. A. Rosen i S. L. Berger. "Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5." Molecular and Cellular Biology 16, nr 2 (luty 1996): 593–602. http://dx.doi.org/10.1128/mcb.16.2.593.
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Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.
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Parthun, M. R., i J. A. Jaehning. "Purification and characterization of the yeast transcriptional activator GAL4." Journal of Biological Chemistry 265, nr 1 (styczeń 1990): 209–13. http://dx.doi.org/10.1016/s0021-9258(19)40217-2.
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Giniger, E., i M. Ptashne. "Cooperative DNA binding of the yeast transcriptional activator GAL4." Proceedings of the National Academy of Sciences 85, nr 2 (1.01.1988): 382–86. http://dx.doi.org/10.1073/pnas.85.2.382.
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