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PubMed2003

FEV acts as a transcriptional repressor through its DNA-binding ETS domain and alanine-rich domain.

Although most Ets transcription factors have been characterized as transcriptional activators, some of them display repressor activity. Here we characterize an Ets-family member, the very specifically expressed human Fifth Ewing Variant (FEV), as a transcriptional repressor. We show that among a broad range of human cell lines, only Dami megakaryocytic cells express FEV. This nuclear protein binds to Ets-binding sites, such as that of the human ICAM-1 promoter. We used this promoter to demonstrate that FEV can repress both basal transcription and, even more strongly, ectopically Ets-activated transcription. We identified two domains responsible for FEV-mediated repression: the ETS domain, responsible for passive repression, and the carboxy-terminal alanine-rich domain, involved in active repression. In the Ets-independent LEXA system also, FEV acts as a transcriptional repressor via its alanine-rich carboxy-terminal domain. The mechanism by which FEV actively represses transcription is currently unknown, since FEV-triggered repression is not reversed by the histone deacetylase inhibitor trichostatin A. We also showed that long-term overexpression of FEV proteins containing the alanine-rich domain prevents cell clones from growing, whereas clones expressing a truncated FEV protein lacking this domain develop like control cells. This confirms the importance of this domain in FEV-triggered repression.

Alanine↗
PubMed1989

Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene.

The expression of the cyanobacterial recA gene, isolated from Anabaena variabilis, has been examined at the levels of transcript and protein abundance. Exposure of the cyanobacterium to a variety of DNA-damaging agents, including mitomycin C, methyl methanesulfonate, and UV irradiation, results in a rapid increase in the abundance of the recA transcript above basal levels as determined by Northern (RNA) blot analysis. A concomitant increase in the abundance of a 37- to 38-kilodalton polypeptide was also detected by Western (immuno-) blot analysis of soluble cyanobacterial polypeptides using polyclonal antiserum directed against the Escherichia coli recA protein. The cyanobacterial polypeptide is of the same molecular mass as that synthesized by an in vitro, DNA-directed procaryotic transcription-translation system primed with an A. variabilis genomic fragment containing the recA gene. Nucleotide sequence analysis of the cyanobacterial gene revealed a protein of 358 amino acids with a molecular weight of 38,403 daltons. The A. variabilis and E. coli recA genes share similarity at 58% of the amino acid residues; however, an E. coli-like lexA repressor-binding site is not present in the A. variabilis promoter region. The similarities of A. variabilis and E. coli recA expression and gene sequence are discussed.

Amino Acid Sequence↗
PubMed1985

Structural and functional organization of the colicin E1 operon.

We analyzed the structural and functional relationships among independently cloned segments of the plasmid ColE1 region that regulates and codes for colicin E1 (cea), immunity (imm), and the mitomycin C-induced lethality function (lys). On the basis of physiological properties, restriction endonuclease mapping, and DNA sequence analysis, the following recombinant plasmids were determined to represent three major functional classes: pNP12 (cea+, imm+, lys+), pNP4 (cea+, imm+, lys-), and pNP6 (cea+, imm-, lys-). Our results have established the order, boundaries, and relative orientation of the three structural genes, the location of the promoter region for imm gene transcription, and the predicted amino acid sequences of the imm and lys gene products. Hydropathicity analysis suggests that both proteins have hydrophobic amino acid segments characteristic of membrane-associated proteins. A model for the structure and expression of the colicin E1 operon is proposed in which the cea and lys genes are expressed from a single inducible promoter that is controlled by the lexA repressor in response to the SOS system of Escherichia coli. The imm gene lies between the cea and lys genes and is expressed by transcription in the opposite direction from a promoter located within the lys gene. This arrangement of structural genes indicates that the transcriptional units for all three genes overlap. We suggest that the formation of anti-sense RNA may be an important element in the coordinate regulation of gene expression in this system.

Amino Acid Sequence↗
PubMed2004

Plant class B HSFs inhibit transcription and exhibit affinity for TFIIB and TBP.

Plant heat shock transcription factors (HSFs) are capable of transcriptional activation (class A HSFs) or both, activation and repression (class B HSFs). However, the details of mechanism still remain unclear. It is likely, that the regulation occurs through interactions of HSFs with general transcription factors (GTFs), as has been described for numerous other transcription factors. Here, we show that class A HSFs may activate transcription through direct contacts with TATA-binding protein (TBP). Class A HSFs can also interact weakly with TFIIB. Conversely, class B HSFs inhibit promoter activity through an active mechanism of repression that involves the C-terminal regulatory region (CTR) of class B HSFs. Deletion analysis revealed two sites in the CTR of soybean GmHSFB1 potentially involved in protein-protein interactions with GTFs: one is the repressor domain (RD) located in the N-terminal half of the CTR, and the other is a TFIIB binding domain (BD) that shows affinity for TFIIB and is located C-terminally from the RD. A Gal4 DNA binding domain-RD fusion repressed activity of LexA-activators, while Gal4-BD proteins synergistically activated strong and weak transcriptional activators. In vitro binding studies were consistent with this pattern of activity since the BD region alone interacted strongly with TFIIB, and the presence of RD had an inhibitory effect on TFIIB binding and transcriptional activation.

Amino Acid Sequence↗
PubMed1985

Evidence for RecA protein association with the cell membrane and for changes in the levels of major outer membrane proteins in SOS-induced Escherichia coli cells.

Membrane fractions from Escherichia coli cells expressing DNA damage-inducible (SOS) functions contain elevated quantities of RecA protein (L. J. Gudas and A. B. Pardee, J. Mol. Biol. 101:459-477, 1976). We used two-dimensional polyacrylamide gel electrophoresis to separate membrane proteins from several strains to determine whether this effect is an artifact due to contamination of membranes during preparation by the large amount of cytoplasmic RecA present in SOS-induced cells. We found that amplification of RecA+ protein without a DNA-damaging treatment does not result in increased RecA-membrane association, whether recA is depressed specifically by an operator-constitutive recA allele or coordinately with other SOS genes by a lexA mutation that inactivates their common repressor. In contrast, large amounts of RecA appear in membrane fractions from undamaged cells of an SOS-constitutive strain carrying recA730, which encodes a spontaneously SOS-activated RecA. We conclude that the increased association of RecA with the membrane fraction requires the presence of the activated form of RecA, and that this association may contribute significantly to the SOS response. We describe also striking effects of SOS expression on the levels of the outer membrane proteins OmpA, OmpC, and OmpF.

Bacterial Outer Membrane Proteins↗
PubMed1994

Transcriptional activation of NFI/CTF1 depends on a sequence motif strongly related to the carboxyterminal domain of RNA polymerase II.

Initiation of RNA polymerase II-directed transcription is mediated by DNA sequence specific activator proteins interacting with components of the basal transcription machinery. NFI/CTF is a family of such binding proteins which have been shown to stimulate transcription via proline-rich activation domains. In order to identify residues crucial for its activator function, a pool of CTF1 mutants was cloned and fused to the bacterial repressor LexA. Transcriptional activation of these constructs was monitored in a Saccharomyces cerevisiae reporter assay. Our studies reveal the existence of a core domain in CTF1 between residues 463 and 508 essential for transcriptional activation functions. It contains the sequence motif SPTSPSYSP, which is strongly related to the heptapeptide repeat YSPTSPS present in the carboxyterminal domain (CTD) of RNA polymerase II. Removal of the entire CTD related motif, as well as substitution of key amino acids therein, abolish CTF1 mediated transcriptional activation.

Amino Acid Sequence↗
PubMed2005

LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator.

The bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes (hoxEFUYH) which are transcribed as one unit. The transcription of the hox-operon is regulated by a promoter situated upstream of hoxE. The transcription start point was located at -168 by 5'Race. Several promoter probe vectors carrying different promoter fragments revealed two regions to be essential for the promoter activity. One is situated in the untranslated 5'leader region and the other is found -569 to -690 nucleotides upstream of the ATG. The region further upstream was shown to bind a protein. Even though an imperfect NtcA binding site was identified, NtcA did not bind to this region. The protein binding to the DNA was purified and found to be LexA by MALDI-TOF. The complete LexA and its DNA binding domain were overexpressed in Escherichia coli. Both were able to bind to two sites in the examined region in band-shift-assays. Accordingly, the hydrogenase activity of a LexA-depleted mutant was reduced. This is the first report on LexA acting not as a repressor but as a transcriptional activator. Furthermore, LexA is the first transcription factor identified so far for the expression of bidirectional hydrogenases in cyanobacteria.

Bacterial Proteins↗
PubMed1991

Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium.

According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass.

Bacterial Proteins↗
PubMed1999

The Tup1-Cyc8 protein complex can shift from a transcriptional co-repressor to a transcriptional co-activator.

Cyc8(Ssn6)-Tup1, a general co-repressor complex, is recruited to promoter DNA via interactions with DNA-binding regulatory proteins and inhibits the transcription of many different yeast genes. Previous studies have established that repression function of the complex is performed by one subunit of the complex, the Tup1 protein, and requires specific components of the RNA polymerase II holoenzyme such as Sin4 and Rgr1. In this study we test the transcriptional activity of the Cyc8 subunit using a lexA operator-containing reporter. We show that a LexA-Cyc8 hybrid stimulates transcription when expressed in a tup1Delta, a sin4Delta, or a rgr1Delta strain, suggesting that transcriptional activation is an intrinsic property of the Cyc8-Tup1 co-repressor. In support of this notion we demonstrate that Cyc8-Tup1 has a dual function on CIT2, a gene encoding a citrate synthase that is expressed upon mitochondrial dysfunction. First, we show that Cyc8-Tup1 is tethered to CIT2 promoter by interacting with the activation domain of Rtg3, a bHLH/L-Zip DNA-binding transactivator of CIT2. Next we demonstrate that Cyc8-Tup1 activates CIT2 transcription in response to mitochondrial dysfunction, and this stimulatory effect is mediated by Cyc8. In contrast, basal (noninduced) expression of this gene is inhibited by Tup1. These findings establish a positive role for the Cyc8-Tup1 complex in transcription and support a model by which specific metabolic signals may convert the Cyc8-Tup1 transcriptional co-repressor to a co-activator of certain promoters.

DNA-Binding Proteins↗
PubMed1996

Structural and functional organization of the Yersinia pestis bacteriocin pesticin gene cluster.

The primary structure of a 2671 bp DNA fragment between the pla gene (encoding plasminogen activator) and the origin of replication of the wild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 1074 and 426 bp with opposite transcription polarities were identified on both strands. They encode a 357 aa pesticin activity protein (Pst) and a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and serve as rho-independent transcription terminator sequences for both genes. The site for the interaction with the LexA repressor of the SOS system was found in another palindromic structure preceding the pst structural gene. A deduced 39.9 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pentapeptide typical of TonB-dependent colicins (TonB box) that is necessary for the interaction with the yersiniabactin/pesticin receptor and for active energy-dependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not significantly influence the bactericidal activity of the truncated pesticin. The pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino acids. A deduced 16 kDa Pim protein has an N-terminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pl. The immunity protein was localized in the periplasmic space and in the outer-membrane fraction after its overexpression under the polymerase T7 promoter. Several other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necessary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (> 70%) identity with other colicin genes. The DNA sequence located 284 bp upstream of the pim gene showed more than 90% similarity to antisense RNA I of the ColE1 replicon. This defined the location of the pYP358 origin of ColE1-type replication.

Amino Acid Sequence↗
PubMed2005

The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism.

Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.

Amino Acid Sequence↗
PubMed2000

Recruitment of the yeast Tup1p-Ssn6p repressor is associated with localized decreases in histone acetylation.

Posttranslational acetylation of histones is an important element of transcriptional regulation. The yeast Tup1p repressor is one of only a few non-enzyme proteins known to interact directly with the amino-terminal tail domains of histones H3 and H4 that are subject to acetylation. We demonstrated previously that Tup1p interacts poorly with more highly acetylated isoforms of these histones in vitro. Here we show that two separate classes of promoters repressed by Tup1p are associated with underacetylated histones in vivo. This decreased histone acetylation is dependent upon Tup1p and its partner Ssn6p and is localized to sequences near the point of Tup1p-Ssn6p recruitment. Increased acetylation of histones H3 and H4 is observed upon activation of these genes, but this increase is not dependent on transcription per se. Direct recruitment of Tup1p-Ssn6p complexes via fusion of Tup1p to the lexA DNA binding domain is sufficient to confer repression and induce decreased acetylation of H3 and H4 at a target promoter. Taken together, our results suggest that stable decreases in histone acetylation levels are directed and/or maintained by the Tup1p-Ssn6p repressor complex.

Acetylation↗
PubMed1982

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli.

Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42 degrees C but not at 30 degrees C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA+ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA+. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.

Bacterial Proteins↗
PubMed1987

Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12.

Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA+ gene product. The requirement of SDR for recA+ can be suppressed by rin mutations (for recA+-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA+-dependent SDR seen in rnh- rin+ lexA+ strains, recA+-independent in rnh- rin- lexA+, and recA+-independent in rnh- rin+ lexA(Def). The expression of SDR in rin- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA+ requirement by rin mutations was shown to depend on some new function of the recF+ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF+. The lexA3 mutation inhibited recA+-dependent SDR via reducing the amount of recA+ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh- rin- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA+-regulated gene product in the recA+-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA+, rin+ and recF+ genes.

DNA Replication↗
PubMed1999

Nrg1 is a transcriptional repressor for glucose repression of STA1 gene expression in Saccharomyces cerevisiae.

Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. The NRG1 gene encodes a 25-kDa C2H2 zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of the NRG1 gene causes a fivefold increase in the level of the STA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in both ssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.

Amino Acid Sequence↗
PubMed1996

A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP.

The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection. This assay, designated the "split-hybrid system," contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.

Amino Acid Sequence↗
PubMed1997

Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family.

We have identified Xbp1 (XhoI site-binding protein 1) as a new DNA-binding protein with homology to the DNA-binding domain of the Saccharomyces cerevisiae cell cycle regulating transcription factors Swi4 and Mbp1. The DNA recognition sequence was determined by random oligonucleotide selection and confirmed by gel retardation and footprint analyses. The consensus binding site of Xbp1, GcCTCGA(G/A)G(C/A)g(a/g), is a palindromic sequence, with an XhoI restriction enzyme recognition site at its center. This Xbpl binding site is similar to Swi4/Swi6 and Mbp1/Swi6 binding sites but shows a clear difference from these elements in one of the central core bases. There are binding sites for Xbp1 in the G1 cyclin promoter (CLN1), but they are distinct from the Swi4/Swi6 binding sites in CLN1, and Xbp1 will not bind to Swi4/Swi6 or Mbp1/Swi6 binding sites. The XBP1 promoter contains several stress-regulated elements, and its expression is induced by heat shock, high osmolarity, oxidative stress, DNA damage, and glucose starvation. When fused to the LexA DNA-binding domain, Xbp1 acts as transcriptional repressor, defining it as the first repressor in the Swi4/Mbp1 family and the first potential negative regulator of transcription induced by stress. Overexpression of XBP1 results in a slow-growth phenotype, lengthening of G1, an increase in cell volume, and a repression of G1 cyclin expression. These observations suggest that Xbp1 may contribute to the repression of specific transcripts and cause a transient cell cycle delay under stress conditions.

Amino Acid Sequence↗