GAL11P: A yeast mutation that potentiates the effect of weak GAL4-derived activators

Himmelfarb, H J; Pearlberg, Joseph; Ptashne, Mark

doi:10.1016/0092-8674(90)90425-e

Cited by 144 publications

(119 citation statements)

References 63 publications

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“…We suggest therefore that GALli protein functions as a basal transcription factor of some genes and that the GAL1l dependency is determined by the promoter context of the genes. This is consistent with the observations that the function of GALlI is pleiotropic (15,16,21,22). Since GALli protein did not bind a specific DNA sequence as judged by footprint analysis (H.S., unpublished results), it may regulate the basal transcription by protein-protein interactions with the basal or general transcription factors.…”

Section: Resultssupporting

confidence: 79%

“…Since GALli protein did not bind a specific DNA sequence as judged by footprint analysis (H.S., unpublished results), it may regulate the basal transcription by protein-protein interactions with the basal or general transcription factors. However, we have not been able to exclude the possibility that the GALli dependency of a gene was determined by species of activators as postulated (16,21,34). Other combinations of activators and promoters should be studied to clarify the connection between GALli and activators.…”

Section: Resultsmentioning

confidence: 99%

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Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

Sakurai

Hiraoka

Fukasawa

1993

Proc. Natl. Acad. Sci. U.S.A.

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The yeast auxiliary transcription factor GAL1l, a candidate for the coactivator, was partially purified from yeast cells, and its function was characterized in a cell-free ranscription system. The partially purified GAL1l protein stimulated basal transcription from the CYCI core promoter by a factor of 4-5 at the step of preinitiation complex formation. GALll protein also enhanced transcription activated by general regulatory factor 1, GAL4-AH, or GAL4-VP16 to the same extent as the basal ranscription. Therefore, the apparent potentiation of the activators by GALll was attributable to the stimulation of basal transcription. The wild-type GALll protein (but not a mutant-type protein) produced in bacteria stimulated transcription as effectively as GAL1l from yeast. These results suggest that GALll functions as a positive cofactor of basal and activator-induced transcription in a cell-free transcription system. that potentiates a weak activator GAL4-AH. Surprisingly, the mutation is a missense mutation within GALI and was therefore designated GALIIP (P stands for potentiator). They suggest a direct interaction between GAL1l and GAL4 molecules and suggest that the complex of the two proteins is a strong activator. They further find that GALII is required for normal functioning of PPR1, an activator for the URA3 gene. Puzzlingly, GALI is also involved in the transcriptional repression of genes, since a loss-of-function mutation of GALII, called sptl3, restores the transcription of genes inactivated by insertion of the yeast transposon Ty (22).To get insight into the complex pleiotrophic functions of GALH in vivo, we analyzed the role of GAL1l biochemically. We have partially purified GALli protein and studied how it works in a cell-free transcription system. We have found that GAL1l enhances the basal transcription as well as the transcription activated by GAL4-AH, GAL4-VP16, or GRF1. These results suggest that, since GAL1l regulates the basal transcription, it is not a bona fide coactivator, but rather it belongs to a distinctive class of basal transcription factors. (15,27). The major start site of GALli mRNA was Abbreviations: UAS, upstream activation sequence; GRF1, general regulatory factor 1; GST, glutathione S-transferase. MATERIALS AND METHODSITo whom reprint requests should be addressed. 8382

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Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

Sakurai

Hiraoka

Fukasawa

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…A number of proteins exhibit genetic or biochemical properties that make them candidates for interacting with the GAL4 DBD. A mutant of one protein GAL11, called GAL11P, potentiates transcriptional activation by proteins containing the GAL4 DNA-binding domain and weak transcriptional activation domains from other transcription factors (28). The positive effect of GAL11P is reversed by a GAL4 mutation (Lys 20 to Glu) in close proximity to the subregion B amino acids.…”

Section: Discussionmentioning

confidence: 99%

Alterations in the GAL4 DNA-binding Domain Can Affect Transcriptional Activation Independent of DNA Binding

Corton

Granell

Johnston

1998

Journal of Biological Chemistry

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The GAL4 protein belongs to a large class of fungal transcriptional activator proteins encoding within their DNA-binding domains (DBD) six cysteines that coordinate two atoms of zinc (the Zn 2 Cys 6 domain). In an effort to characterize the interactions between the Zn 2 Cys 6 class transcriptional activator proteins and their DNAbinding sites, we have replaced in the full-length GAL4 protein small regions of the Zn 2 Cys 6 domain with the analogous regions of another Zn 2 Cys 6 protein called PPR1 an activator of pyrimidine biosynthetic genes. Alterations between the first and third cysteines abolished binding to GAL4 (upstream activation sequence of GAL (UAS G )) or PPR1 (upstream acitvation sequence of UAS) DNA-binding sites and severely reduced transcriptional activation in yeast. In contrast, alterations between the third and fourth cysteines had only minor effects on binding to UAS G but led to substantial decreases in activation in both yeast and a mammalian cell line. In the crystal structure of the GAL4 DBD-UAS G complex (Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S. C. (1992) Nature 356, 408 -414), this region is facing away from the DNA, making it likely that there exists within the GAL4 DBD an accessible domain important in activation.The GAL4 protein is an 881-amino acid transcriptional activator of the GAL and MEL1 genes in Saccharomyces cerevisiae. The mechanism by which GAL4 activates transcription is conserved because GAL4 also activates transcription in plant, Drosophila, and mammalian cells if GAL4 DNA-binding sites (UAS G ) 1 are present in the promoter of appropriate reporter genes (reviewed in Ref. 1). GAL4 is a member of a large family of fungal transcriptional activators that contain within their DNA-binding domains a conserved cysteine-rich region encoding six cysteine residues that coordinate two atoms of zinc (the Zn 2 Cys 6 domain). The DNA recognition sequences to which these activators bind have been identified and all have in common a CGG or related triplet in each of the two symmetrically opposed half-sites (2-4). The sequences between the triplets as well as the spacing of the triplet sequence are highly variable, indicating that these variables may in large part determine specific recognition. Based on the extent of homology between the Zn 2 Cys 6 activators, it was hypothesized that the Cys-rich region performs a function common to all members of this class of proteins and the region immediately adjacent, which encodes divergent sequences, determines DNA binding specificity (5). We previously demonstrated that this model is essentially correct (6). All but one (Lys 23 ) of the 28 amino acids in the Zn 2 Cys 6 region of GAL4 can be replaced by the analogous sequences of PPR1, an activator of pyrimidine biosynthetic genes (7), without changing DNA binding specificity. In contrast, replacing the 14 amino acids immediately adjacent to the Zn 2 Cys 6 region resulted in switching the DNA binding specificity to that of PPR1. Similar results were obtained when hybrids of L...

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“…Experiments performed in eukaryotic cells have also shown that transcription can be activated by the direct fusion of DNA-binding domains to various components of the transcriptional machinery. For example, fusion of the E. coli LexA repressor, a sequence-specific DNAbinding protein, to the wild-type Gal11 protein creates a powerful transcriptional activator in yeast that works on promoters bearing LexA-binding sites, and this activation depends on the portion of Gal11 that mediates its association with the RNAP II holoenzyme (Himmelfarb et al 1990;Barberis et al 1995; see also Farrell et al 1996;Gaudreau et al 1997). Similarly, direct or indirect fusion of a DNA-binding domain to the yeast TATAbinding protein (TBP) creates a transcriptional activator that works on promoters bearing a recognition site for the DNA-binding domain upstream of the TATA ele- …”

Section: Transcriptional Activation In E Coli By Tethering a Subunitmentioning

confidence: 99%

“…For this purpose we took advantage of a pair of protein domains originally shown to interact in yeast cells. Transcriptional activation in yeast can be triggered by an apparently fortuitous interaction between the dimerization region of the yeast transcriptional activator Gal4 and a mutant form of the Gal11 protein (Himmelfarb et al 1990;Barberis et al 1995), which despite its name, is a component of the RNAP II holoenzyme and is required for full transcription of many genes (Kim et al 1994;Barberis et al 1995;Hengartner et al 1995). Ordinarily, in yeast, the dimerization …”

mentioning

confidence: 99%

Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target

Dove¹,

Hochschild²

1998

Genes & Development

148

136

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Evidence obtained in both eukaryotes and prokaryotes indicates that arbitrary contacts between DNA-bound proteins and components of the transcriptional machinery can activate transcription. Here we demonstrate that the Escherichia coli protein, which copurifies with RNA polymerase, can function as a transcriptional activator when linked covalently to a DNA-binding protein. We show further that can function as an activation target when this covalent linkage is replaced by a pair of interacting polypeptides fused to the DNA-binding protein and to , respectively. Our findings imply that the protein is associated with RNA polymerase holoenzyme in vivo, and provide support for the hypothesis that contact between a DNA-bound protein and any component of E. coli RNA polymerase can activate transcription.[Key Words: subunit; E. coli; RNA polymerase; transcriptional activator] Received December 3, 1997; revised version accepted January 15, 1998. Recent findings in both eukaryotes and prokaryotes indicate that arbitrary protein-protein contacts can trigger gene activation provided one of the protein partners is tethered to the DNA and the other is a component (or is tethered to a component) of RNA polymerase (RNAP) (Barberis et al. 1995;Chatterjee and Struhl 1995;Klages and Strubin 1995;Xiao et al. 1995;Apone et al. 1996;Farrell et al. 1996;Dove et al. 1997;Gaudreau et al. 1997;Gonzalez-Couto et al. 1997;Lee and Struhl 1997; for review, see Ptashne and Gann 1997). Experiments in yeast have shown further that direct fusion of a DNA-binding domain to a component of the RNAP II holoenzyme can activate transcription from a promoter bearing a recognition site for the DNA-binding domain (Barberis et al. 1995;Farrell et al. 1996;Gaudreau et al. 1997; for review, see Ptashne and Gann 1997), but analogous experiments have not been performed previously in bacteria.RNAP in Escherichia coli consists of an enzymatic core composed of subunits ␣, ␤, and ␤Ј in the stoichiometry ␣ 2 ␤␤Ј, and one of several alternative factors that confer on the enzyme the ability to recognize specific promoters (Burgess 1976;Hellman and Chamberlin 1988). An additional protein, omega ( ), has been called a subunit of RNAP on the basis of its copurification with RNAP core and holoenzyme in near stoichiometric amounts (Burgess 1969). The function of is unknown and, unlike the other subunits, is not required for transcription either in vitro (Heil and Zillig 1970) or in vivo (Gentry and Burgess 1989;Gentry et al. 1991). Cells deleted for the gene encoding (rpoZ) have no discernible mutant phenotypes (Gentry and Burgess 1989;Gentry et al. 1991).Many natural activators in bacteria bind the DNA near the promoters they regulate and interact directly with one or more subunits of RNAP (Busby and Ebright 1994). The best known target of these interactions is the ␣ subunit of RNAP (Ishihama 1992;Russo and Silhavy 1992;Ebright and Busby 1995;Niu et al. 1996). Some activators, however, interact with the subunit (Hochschild 1994;Kuldell and Hochschild 1994;Li et al. 1...

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GAL11P: A yeast mutation that potentiates the effect of weak GAL4-derived activators

Cited by 144 publications

References 63 publications

Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

Alterations in the GAL4 DNA-binding Domain Can Affect Transcriptional Activation Independent of DNA Binding

Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target

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