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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The promoters of the Saccharomyces cerevisiae histone H3 and H4 genes were examined for cis-acting DNA sequence elements regulating transcription and cell division cycle control. Deletion and linker disruption mutations identified two classes of regulatory elements: multiple cell cycle activation (CCA) sites and a negative regulatory site (NRS). Duplicate 19-bp CCA sites are present in both the copy I and copy II histone H3-H4 promoters arranged as inverted repeats separated by 45 and 68 bp. The CCA sites are both necessary and sufficient to activate transcription under cell division cycle control. A single CCA site provides cell cycle control but is a weak transcriptional activator, while an inverted repeat comprising two CCA sites provides both strong transcriptional activation and cell division cycle control. The NRS was identified in the copy I histone H3-H4 promoter. Deletion or disruption of the NRS increased the level of the histone H3 promoter activity but did not alter the cell division cycle periodicity of transcription. When the CCA sites were deleted from the histone promoter, the NRS element was unable to confer cell division cycle control on the remaining basal level of transcription. When the NRS element was inserted into the promoter of a foreign reporter gene, transcription was constitutively repressed and did not acquire cell cycle regulation.
Mol Cell Biol 1992 Dec
PMID:Histone H3 transcription in Saccharomyces cerevisiae is controlled by multiple cell cycle activation sites and a constitutive negative regulatory element. 144 78

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.
Mol Cell Biol 1992 Dec
PMID:The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding. 144 98

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.
Mol Cell Biol 1992 Dec
PMID:Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases. 144 7

We have previously shown that transcription of the Xenopus U6 snRNA gene by RNA polymerase III is stimulated in injected Xenopus oocytes by an activator element termed the DSE, which contains an octamer sequence. Data presented here reveal that the DSE contains, in addition, a GC-rich sequence capable of binding Sp1. Both elements are required to obtain wild-type levels of U6 transcription in vivo. The Xenopus U6 DSE exhibits optimal activation properties only when positioned at its normal location upstream from the start site. The U6 Sp1 motif binds the mammalian Sp1 transcriptional activator independently of the Oct-1 protein in vitro. Those mutations that lead to a reduced transcription level in vivo abolish the binding of Sp1 in vitro. Thus, transcriptional stimulation through the Xenopus U6 Sp1 motif is likely to be mediated by a protein with DNA-binding specificity identical to mammalian Sp1. These findings support the notion that RNA polymerase II and III transcription complexes share transactivators.
J Mol Biol 1992 Nov 20
PMID:A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III. 145 50

The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.
Mol Endocrinol 1992 Jul
PMID:Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast. 150 20

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.
J Mol Endocrinol 1992 Aug
PMID:The function of conserved elements in the promoter of the mouse angiotensinogen gene. 151 23

The c-myb protooncogene, which is preferentially expressed in hematopoietic cells at the G1/S boundary of the cell cycle, encodes a transcriptional activator that functions via DNA binding. The regulatory mechanisms governing this specific pattern of expression are not fully understood, although human c-myb expression appears to be positively autoregulated via myb-binding sites in the 5'-flanking region of the c-myb gene (Nicolaides, N. C., Gualdi, R., Casadevall, C., Manzella, L., and Calabretta, B. (1991) Mol. Cell. Biol. 11, 6166-6176). To determine the contribution of other transcription regulators such as JUN family members in the control of c-myb expression, transient expression assays were carried out which revealed a 6- to a 15-fold enhancement by c-Jun and JunD, but not JunB, in chloramphenicol acetyltransferase reporter gene expression driven by different segments of the human c-myb 5'-flanking region. An Ap1-like element located at nucleotide -149 from the c-myb initiation site appears to be required for this transactivation upon binding to a nuclear protein complex containing c-Jun and JunD, since site-directed mutations of this Ap1-like element abolished c-Jun and JunD binding and transactivation. Exposure of phytohemagglutinin-stimulated peripheral blood mononuclear cells to c-jun and junD antisense oligodeoxynucleotides resulted in a 46 and 43% inhibition of T-lymphocyte proliferation that was accompanied by a decrease in c-myb mRNA levels as compared with sense-treated cultures. Because T-lymphocytes induced to proliferate express c-jun and junD before c-myb, these data suggest a mechanism whereby c-Jun and JunD contribute to the transcriptional activation of c-myb that, in turn, is maintained at the G1/S transition and during S phase by positive autoregulation.
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PMID:The Jun family members, c-Jun and JunD, transactivate the human c-myb promoter via an Ap1-like element. 152 86

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.
Mol Cell Biol 1992 Apr
PMID:Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa. 153 30

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.
Mol Cell Biol 1992 Mar
PMID:Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor. 154 86

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.
Mol Cell Biol 1992 Apr
PMID:ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230. 154 8


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