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Results for “LexA Repressor Protein”

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PubMed2001

Establishment of Polycomb silencing requires a transient interaction between PC and ESC.

Two distinct types of Polycomb complexes have been identified in flies and in vertebrates, one containing ESC and one containing PC. Using LexA fusions, we show that PC and ESC can establish silencing of a reporter gene but that each requires the presence of the other. In early embryonic extracts, we find PC transiently associated with ESC in a complex that includes EZ, PHO, PH, GAGA, and RPD3 but not PSC. In older embryos, PC is found in a complex including PH, PSC, GAGA, and RPD3, whereas ESC is in a separate complex including EZ, PHO, and RPD3.

Animals↗
PubMed1997

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.

Adenosine Triphosphatases↗
PubMed2003

An NMR study on the interaction of Escherichia coli DinI with RecA-ssDNA complexes.

The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA. Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA. On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA- ATPgammaS, but had a slight effect on the structure of ssDNA-RecA-ATPgammaS complexes involving 8mer and 12mer ssDNA. We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA-RecA-ATPgammaS complex, together function to inhibit the co-protease activity of RecA.

Adenosine Triphosphate↗
PubMedDate not supplied

[Minor promoters of phage phi X174 are controlled by CRP-cAMP, lexA, glnG and several other common common regulatory systems of the host cell].

It was found that CRP-cAMP-recognized sequences in DNA being suggested as GTGN7-11CAC (with variability both in domain's structures and in spacer's length) are located non-randomly in promoters. In CRP-cAMP-stimulated promoters they lie upstream the "-35" box and are separated from it by a whole number of DNA turns, whereas in CRP-cAMP-repressed ones they are located downstream "-35" in a half-whole-turn-number distance. Several CRP-, SOS- and NR1-sites in the phi X174 DNA sequence were found and a few new promoters were deduced from it. PCRP1 lies within gene F and has both CRR and ntrC sites and one SOS-operator, PCRP3 (in gene A) has a CRP site which overlaps with the SOS-operator, PA and PCRP2 (in gene G) have sCRP and PD has a stringent discriminator. Four promotors, PCRP1, PCRP2, PA and PB are cloned in the pBR322 plasmid. For cloned PCRP1 the activation by exogenous cAMP and the SOS-induction by the mitomycin C were observed in vivo in pVYB215-containing cells by increasing the levels of beta-lactamase up to 27-fold. The new gene L of the phi X174 is deduced from the DNA sequence. It has two start points, overlaps the gene F inside it and codes for peptides 23 or 19 amino acids in length. These lethal peptides have strong homology in sequence to the cellular protein sulA(sfiA) of E. coli, and L* can cause observed filamentation and death of pVYB215- bearing cells after PCRP1 induction. In the A and A* protein sequences two domains "helix-turn-helix" were found that are homologous to those in CRP and repressors; this makes possible the competition between A* and CRT for its DNA sites that also have some homology. The model of the phi X174 infection cycle control and mechanisms of DNA recognition by CRP-CAMP are discussed. PCRP1 is the first promotor controlled by both three global regulons of E. coli cell.

Bacteriophage phi X 174↗
PubMed1992

Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence.

The Bacillus subtilis dinR gene product is homologous to the LexA protein of Escherichia coli and regulates the expression of dinR and dinC. Using transcriptional fusions in the dinR and the recA genes, we have investigated the epistatic relationship between these two genes during the SOS response induced either by DNA damage or by competence. The results show that after DNA damage, induction of the expression of both recA and dinR is dependent on the activity of the DinR and RecA proteins. A RecA-dependent activity on DinR is proposed as the initial event in the induction of the SOS network. In contrast, the competence-related induction of dinR and recA appears to involve two distinct mechanisms. While one mechanism corresponds to the classical regulation of the SOS response, the other appears to involve an activating factor. Moreover, this factor is active in cells in which competence is prevented by a mutation in the regulatory gene comA.

Bacillus subtilis↗
PubMed1997

The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant.

Escherichia coli DNA polymerase II (Pol II) is a member of the group B, "alpha-like" family of DNA polymerases. Pol II is encoded by the damage-inducible dinA gene and exhibits SOS induction under the control of Lex A repressor. The polB gene was originally designated as the structural gene for Pol II based on the absence of detectable Pol II activity in cell lysates prepared from a strain containing the mutant polB100 allele. Because polB and dinA mapped at different chromosomal locations, it remained an open question whether polB, in addition to lexA, might be involved in regulating the expression of Pol II. We have cloned and sequenced the polB100 mutant allele, including adjacent surrounding sequences, and have expressed the mutant dinA gene from Pol B100 on a high copy number plasmid. Our sequence data reveal that polB and dinA represent the same gene and that the original transduction mapping of polB was inaccurate. We purified the mutant Pol B100 polymerase and show that it retains 5 to 10% of the wild-type level of polymerase activity. The Pol B100 mutation, Gly401 --> Asp401, is not located within any of the five conserved domains that define group B polymerases. Pol B100 retains a wild-type level of 3' --> 5' exonuclease activity. We suggest that the normal level of exonucleolytic proofreading associated with the mutant Pol B100 enzyme may explain the repeated failures, over the past two decades, to detect phenotypes in polB mutant strains.

Bacterial Proteins↗
PubMed1997

Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Krüppel-type zinc finger factor.

The so-called KRAB domain, which is present in about one third of the vertebrate Kruppel-type zinc finger factors, has previously been shown to inhibit transcription in cis when tethered to promoter regions. Here we analyze this effect with fusions of the KRAB domain derived from KOX1/ZNF10 zinc finger protein to the heterologous DNA binding domains of both LexA and GAL4 factors. In transfected human cells, repression of reporter gene transcription is observed not only from proximal promoter positions, but also when KRAB is tethered to DNA at a remote position more than 1.8 kb downstream of the initiation site of transcription. Furthermore, KRAB-mediated silencing over short and long distances is not restricted to RNA polymerase II, since transcription by RNA polymerase III is also repressed. However, transcription by RNA polymerase I and by phage T7 RNA polymerase in mammalian cells are not significantly influenced by the KRAB domain. These latter results may indicate that repression by the KRAB domain, at least under our assay conditions, involves specific inhibition of some component(s) of RNA polymerase II and III transcription, rather than inducing some gross physical alteration of template chromatin structure.

Binding Sites↗
PubMed1997

The minimal transactivation region of Saccharomyces cerevisiae Gln3p is localized to 13 amino acids.

Regulated nitrogen catabolic gene transcription in Saccharomyces cerevisiae is mediated by four positive (Gln3p and Gat1p/Nil1p) and negative (Dal80p/Uga43p and Deh1p/Nil2p/GZF3p) regulators which function in opposition to one another. All four proteins contain GATA-type zinc finger domains, and three of them (Gln3p, Dal80p, and Deh1p) have been shown to bind to GATA sequences situated upstream of genes whose expression is sensitive to nitrogen catabolite repression (NCR). The positive regulators, Gln3p and Gat1p, are able to support transcriptional activation when tethered by LexAp to the promoter of a reporter gene whose upstream activation sequences have been replaced with one or more lexA operator sites. Existing data suggest that these four proteins regulate transcription by competing with one another for binding to the GATA sequences which mediate NCR-sensitive gene expression. We show that the minimal Gln3p domain mediating transcriptional activation consists of 13 amino acids with a predicted propensity to form an alpha-helix. Genetic analysis of this region (Gln3p residues 126 to 138, QQNGEIAQLWDFN) demonstrated that alanine may be substituted for the aromatic and acidic amino acids without destroying transcriptional activation potential. Similar substitution of alanine for the two hydrophobic amino acids, isoleucine and leucine, however, destroys activation, as does introduction of basic amino acids in place of the acidic residues or introduction of proline into the center of the sequence. A point mutation in the Gln3p activation region destroys its in vivo ability to support NCR-sensitive DAL5 expression. We find no convincing evidence that NCR regulates Gln3p function by modulating the functioning of its activation region.

Amino Acid Sequence↗
PubMed1996

Transformation by the Bmi-1 oncoprotein correlates with its subnuclear localization but not its transcriptional suppression activity.

The bmi-1 oncogene cooperates with c-myc in transgenic mice, resulting in accelerated lymphoma development. Altering the expression of Bmi-1 affects normal embryogenesis. The protein product of bmi-1 is homologous to certain Drosophila Polycomb group proteins that regulate homeotic gene expression through alteration of chromatin structure. Chimeric LexA-Bmi-1 protein has previously been shown to repress transcription. How Bmi-1 functions in embryogenesis and whether this relates to the ability of Bmi-1 to mediate cellular transformation is unknown. We demonstrate here that Bmi-1 is able to transform rodent fibroblasts in vitro, providing a system that has allowed us to correlate its molecular properties with its ability to transform cells. We map functional domains of Bmi-1 involved in transcriptional suppression by using the GAL4 chimeric transcriptional regulator system. Deletion analysis shows that the centrally located helix-turn-helix-turn-helix-turn (HTHTHT) motif is necessary for transcriptional suppression whereas the N-terminal RING finger domain is not required. We demonstrate that nuclear localization requires KRMK (residues 230 to 233) and that the absence of nuclear entry ablates transformation. In addition, we find that the subnuclear localization of wild-type Bmi-1 to the rim of the nucleus requires the RING finger domain and correlates with its ability to transform. Our studies with Bmi-1 deletion mutants suggest that the ability of Bmi-1 to mediate cellular transformation correlates with its unique subnuclear localization but not its transcriptional suppression activity.

Animals↗