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Query: UNIPROT:P06889 (Mol)
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We have carried out deletion analyses of a tobacco transcription activator, TGA1a, in order to define its functional domains. TGA1a belongs to the basic-region-leucine zipper (bZIP) class of DNA-binding proteins. Like other proteins of this class, it binds to its target DNA as a dimer, and its bZIP domain is necessary and sufficient for specific DNA binding. A mutant polypeptide containing the bZIP domain alone, however, shows a lower DNA-binding affinity than the full-length TGA1a. The C-terminal portion of TGA1a, which is essential for the higher DNA-binding affinity, contains a polypeptide region that can stabilize dimeric forms of the protein. This polypeptide region is designated the dimer stabilization (DS) region. Under our in vitro conditions, TGA1a derivatives with the DS region and those without the region do not form a detectable mixed dimer. This result indicates that in addition to the leucine zipper, the DS region can serve as another determinant of the dimerization specificity of TGA1a. In fact, the DS region, when fused to another bZIP protein, C/EBP, can inhibit dimer formation between the fusion protein and native C/EBP, whereas each of these can form homodimers. Such a portable determinant of dimerization specificity has potential application in studies of DNA-binding proteins as well as in biotechnology.
Mol Cell Biol 1992 Nov
PMID:Identification of a novel dimer stabilization region in a plant bZIP transcription activator. 140 63

CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa. CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1. A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA. However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding. Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly. The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N. crassa. Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency. However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1. The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements. The basic region was required for DNA binding but not for dimerization. CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4. When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented. DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization. Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.
Mol Cell Biol 1991 Feb
PMID:Characterization of Neurospora CPC1, a bZIP DNA-binding protein that does not require aligned heptad leucines for dimerization. 182 60

We have isolated and characterized a tobacco gene, designated G13, encoding a leucine zipper DNA-binding protein related to the transcription activator TGA1a. The G13 coding region is divided into eight exons and the amino acid sequence of the encoded protein (PG13) shows 76% homology to TGA1a. Their putative DNA-contacting regions (basic domains) are identical and they both bind to the same target sequences in vitro. By contrast, some differences are apparent between these proteins at the carboxyl end of the dimerization region (leucine zipper). The basic and leucine zipper domains are encoded on separate small exons. Analysis by DNAse I footprinting, gel shift and competition experiments revealed that TGA1a and PG13 synthesized in Escherichia coli, and the tobacco nuclear factor ASF-1 all bind to at least one site in the 5' upstream region of G13. The presence of a TGA1a binding site in the upstream region of a TGA1a-related gene suggests that transcription of this gene is autoregulated.
Mol Gen Genet 1991 Oct
PMID:The tobacco transcription activator TGA1a binds to a sequence in the 5' upstream region of a gene encoding a TGA1a-related protein. 192 69

nur77 is a growth factor-inducible immediate early gene that encodes a protein with extensive sequence homology to members of the steroid/thyroid hormone receptor superfamily. By analogy to steroid receptors, the Nur77 protein is thought to act as a ligand-dependent transcription factor that regulates the genomic response to growth factors. Using chimeric gene constructs, we show that Nur77 can indeed function as a transcription activator. Furthermore, Nur77 chimeras can activate transcription in the absence of an exogenously added ligand.
Mol Endocrinol 1991 Jun
PMID:Transcriptional activation by Nur77, a growth factor-inducible member of the steroid hormone receptor superfamily. 192 99

The biosynthesis of methionine in Escherichia coli is under complex regulation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S-adenosyl-methionine which functions as a corepressor for the MetJ protein. Recently, a new regulatory locus, metR, has been identified. The MetR protein is required for both metE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA-binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metabolic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metE gene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group.
Mol Microbiol 1991 Jul
PMID:Regulation of methionine synthesis in Escherichia coli. 194 95

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.
Mol Cell Biol 1990 Oct
PMID:The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1. 211 90

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.
Mol Cell Biol 1990 Nov
PMID:Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes. 212 31

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.
Mol Cell Biol 1990 Apr
PMID:Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites. 218 Dec 74

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.
Mol Cell Biol 1990 Jun
PMID:Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans. 218 10

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.
Mol Cell Biol 1989 Nov
PMID:Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element. 255 42


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