Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “LexA Repressor Protein”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources
PubMed1987

Dual level control of the Escherichia coli pheST-himA operon expression. tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli have established that the pheST operon transcription is controlled by a Phe-tRNA(Phe)-mediated attenuation mechanism. More recently, the himA gene, encoding the alpha-subunit of integration host factor, was recognized immediately downstream from pheT, possibly forming part of the same transcriptional unit. By using the in-vitro transcription and S1 mapping techniques, transcription termination after pheT could be excluded, indicating that himA can be expressed from polycistronic messenger RNAs encompassing the pheST region. However, the presence of a secondary promoter able to express himA and located within pheT is demonstrated. To further investigate the regulation of the pheST-himA operon expression, genetic fusions between various parts of this operon and the lacZ gene were constructed and studied. Our results confirm the autoregulation of himA previously described, and demonstrate that it occurs through the modulation of the secondary promoter activity within pheT. Surprisingly, it is found that the pheST promoter is also submitted to the same control. Consistent with this, DNA sequences homologous to the integration host factor binding site consensus are present at the level of both promoters. However, evidence in favor of two different repressor complexes is provided. Previously observed SOS induction of the himA expression is shown to occur through the modulation of both promoter activities. Contrasting with the other genes under SOS control, the LexA protein binding site consensus sequence could not be found in the two promoter regions. This suggests that either the LexA protein directly participates in the formation of an active holorepressor, or that the product of an SOS gene is able to inhibit the formation or the binding of such a repressor. Finally, our results indicate that the pheST-himA operon expression is controlled by two different mechanisms acting independently. (1) The phenylalanyl-tRNA synthetase and the himA product expressions are controlled by an operator-repressor type mechanism, in which the himA product and the SOS network are involved. (2) Through its partial cotranscription with pheST, himA expression is also under attenuation control. The latter control may provide a way to couple the intracellular concentration of the himA product to the functional state of the translational apparatus.

DNA Repair↗
PubMed1987

Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.

DNA Repair↗
PubMed2000

Roles for the Saccharomyces cerevisiae SDS3, CBK1 and HYM1 genes in transcriptional repression by SIN3.

The Saccharomyces cerevisiae Sin3 transcriptional repressor is part of a large multiprotein complex that includes the Rpd3 histone deacetylase. A LexA-Sin3 fusion protein represses transcription of promoters with LexA binding sites. To identify genes involved in repression by Sin3, we conducted a screen for mutations that reduce repression by LexA-Sin3. One of the mutations identified that reduces LexA-Sin3 repression is in the RPD3 gene, consistent with the known roles of Rpd3 in transcriptional repression. Mutations in CBK1 and HYM1 reduce repression by LexA-Sin3 and also cause defects in cell separation and altered colony morphology. cbk1 and hym1 mutations affect some but not all genes regulated by SIN3 and RPD3, but the effect on transcription is much weaker. Genetic analysis suggests that CBK1 and HYM1 function in the same pathway, but this genetic pathway is separable from that of SIN3 and RPD3. The remaining gene from this screen described in this report is SDS3, previously identified in a screen for mutations that increase silencing at HML, HMR, and telomere-linked genes, a phenotype also seen in sin3 and rpd3 mutants. Genetic analysis demonstrates that SDS3 functions in the same genetic pathway as SIN3 and RPD3, and coimmunoprecipitation experiments show that Sds3 is physically present in the Sin3 complex.

Base Sequence↗
PubMed1988

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells.

UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.

DNA Repair↗
PubMed1990

Cloning, sequence and expression in Escherichia coli of the Methylobacillus flagellatum recA gene.

By means of interspecific complementation of an Escherichia coli recA- mutation with phasmids containing a gene bank from an obligate methylotroph, Methylobacillus flagellatum (Mf), the recA+ gene from this bacterium was identified. When expressed in an E. coli recA- host, it can function in recombination, DNA repair, and prophage induction. The nucleotide sequence of the gene has been determined. The coding region consists of 1032 bp specifying 344 amino acids. The deduced RecA protein structure shows a striking homology with RecA from other bacteria, except for the C-terminal region and some residues which were proposed to be responsible for the coprotease ability of RecA proteins. The region preceding the recA-Mf gene start codon has no SOS box--the LexA repressor binding site. Expression of the recA-Mf gene in E. coli proved to be DNA-damage independent.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of Gluconobacter oxydans recA gene and its inhibitory effect on the function of the host wild-type recA gene.

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.

Amino Acid Sequence↗
PubMed2026

Mapping Protein Occupancy on DNA with an Unnatural Cytosine Modification.

The epigenome provides a dynamic layer of gene regulatory control above the static genetic sequence. DNA base modifications are key epigenetic regulators, predominantly found within CpG contexts in mammalian genomes. Working in tandem with these DNA modifications, chromatin-associated proteins and transcription factors further control gene expression. Given the interplay of these factors, concurrent mapping of DNA base modifications with protein-DNA occupancy can greatly aid in interpreting the epigenome. Existing multimodal mapping methods include the use of DNA methyltransferases to mark accessible, protein-unbound DNA in non-CpG contexts. However, such approaches can either confound readouts with native DNA modifications or constrain users to third-generation sequencing approaches. To circumvent these limitations, we explored the possibility of introducing an unnatural DNA base modification, 5-carboxymethylcytosine, as an alternative label for protein occupancy. Here, we report our efforts to rationally engineer non-CpG-specific DNA methyltransferases to take on neomorphic DNA carboxymethyltransferase (CxMTase) activities. We find that DNA carboxymethylation of cytosines in GpC contexts shows broad compatibility with the most widely used epigenetic detection methods and can be used to reliably report on protein occupancy states. Using this approach, we reveal the single-molecule binding patterns of LexA, a master repressor in the bacterial DNA damage (SOS) response, at its self-regulated and endogenously methylated promoter. We thus show that unnatural DNA modifications can uncover novel biological insights and potentiate new approaches to multimodal epigenetic profiling.

DNA↗
PubMed1996

SIN3-dependent transcriptional repression by interaction with the Mad1 DNA-binding protein.

The SIN3 gene in Saccharomyces cerevisiae encodes a negative regulator of transcription of a large number of genes. Mouse homologs of SIN3 have been identified through screens for proteins interacting with the mammalian Mad1 protein, a transcriptional repressor. We find that yeast Sin3 (ySin3) interacts with Madl and that, as for mouse Sin3, the N terminus of Mad1 interacts with the PAH2 domain of ySin3. Although Mad1 (a basic helix-loop-helix leucine zipper [bHLH-Zip) protein) forms a heterodimer with the Max bHLH-Zip protein, LexA-Mad1 and VP16-Max do not activate transcription of a reporter gene in a two-hybrid assay. This failure in activation is due to direct repression by ySin3, as LexA-Mad1 and VP16-Max are able to activate the two-hybrid reporter in a sin3 mutant. This inhibition of activation by LexA-Mad1 and VP16-Max requires the PAH2 domain of ySin3 and the N-terminal interaction region of Mad1. These data demonstrate that ySin3 functions as a transcriptional repressor by being brought to promoters by interacting with proteins bound to DNA.

Animals↗
PubMed1993

LEF-1 contains an activation domain that stimulates transcription only in a specific context of factor-binding sites.

Lymphoid enhancer factor 1 (LEF-1) is a member of the high mobility group (HMG) family of proteins and participates in the regulation of the T cell receptor (TCR) alpha enhancer. We have previously shown that DNA binding by the HMG domain of LEF-1 induces a sharp bend in the DNA helix. Together with the dependence of LEF-1 on other factor-binding sites to regulate gene expression, DNA bending induced by the HMG domain suggested an 'architectural' role for LEF-1. In this study, we performed experiments to distinguish between a model in which the HMG domain is the only functional determinant of LEF-1 and a model in which additional domains of LEF-1 are involved in the regulation of gene expression. First, we show that the HMG domain alone is not sufficient to stimulate TCR alpha enhancer function. Second, we replaced the HMG domain of LEF-1 with the DNA-binding domain of the bacterial repressor LexA, which binds a specific nucleotide sequence without inducing a sharp bend in the DNA helix. The chimeric LEF-LexA protein increased the activity of a TCR alpha enhancer in which the LEF-1-binding site had been replaced with a LexA recognition sequence. Transcriptional stimulation by LEF-LexA, however, was less efficient than that observed with endogenous LEF-1. The LEF-LexA-mediated activation of gene expression was dependent upon an amino-terminal region of LEF-1 and a specific context of factor-binding sites in the TCR alpha enhancer. Neither multimerized LexA-binding sites, nor TCR alpha enhancers with altered spatial arrangements of factor-binding sites, were functional for regulation by LEF-LexA. Together, these data suggest that an aminoterminal region in LEF-1 contributes to the context-dependent regulation of the TCR alpha enhancer by LEF-1, presumably by interacting with other enhancer-bound proteins.

3T3 Cells↗
PubMed1985

Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12.

We isolated recA mutants with altered protease activity and then examined recombinase activity to determine whether the protease and recombinase functions of the RecA protein of Escherichia coli are separable. We found five mutants that had moderately strong constitutive RecA protease activity but no recombinase activity above the delta recA strain background, the first clear-cut examples of mutants of this class, designated Prtc Rec-. We also isolated 65 mutants that were protease-defective toward the LexA repressor and found that all of them were also recombinase deficient. Four of these mutants retained both partial recombinase activity and partial inducible protease activity. The recombinase-defective mutants were much more sensitive than the recA+ strain to crystal violet, kanamycin, and chloramphenicol, indicating altered membrane permeability. The recA (Prtc Rec-) mutants had a subtle alteration in protease specificity, all being defective in spontaneous induction of phages lambda imm434 and 21. They differed from Prtc Rec+ mutants of comparable or even weaker constitutive protease strength, all of which showed dramatic spontaneous induction of these prophages. However, treating a Prtc Rec- mutant with mitomycin C resulted in significant prophage induction. Thus, the RecA proteins of the Prtc Rec- mutants have constitutive protease activity toward the LexA repressor, but have only DNA damage-activable protease activity toward phage repressors. UV-induced mutagenesis from his to his+ was studied for one Prtc Rec- mutant, and induced mutation frequencies as high as those for the recA+ strain were found despite the absence of recombinase activity.

Antibiotics, Antineoplastic↗
PubMed1983

On the nature of the RecBC and RecF pathways of conjugal recombination in Escherichia coli.

The molecular mechanisms of the RecBC and RecF pathways for genetic recombination in E. coli were investigated by studying the kinetics of RecA protein function during conjugation. RecF recombination in recBC sbcB mutants is shown to be a much slower process than RecBC recombination in recBC+ sbcB+ strains, and is blocked by a mutation in lexA that prevents induction of RecA protein. Progress of the RecF pathway is greatly accelerated by a recAoc mutation which increases synthesis of RecA protein, but this does not restore recombination proficiency to a recBC sbcB lexA mutant. These results are interpreted to suggest that the RecF pathway directs integration of single-stranded Hfr DNA into the recipient chromosome whereas the RecBC pathway catalyses the exchange of largely double stranded DNA. This is consistent with the known stoichiometry of RecA protein catalysed heteroduplex DNA formation in vitro and with the delayed replication of RecF pathway recombinants which approximates to the time required for one round of DNA replication to generate homoduplex DNA. The regulation of the RecF pathway by lexA repressor is discussed in relation to the factors that govern the relative utilization of the two recombination pathways in wild-type cells.

Bacterial Proteins↗
PubMed1986

An inhibitor of SOS induction, specified by a plasmid locus in Escherichia coli.

Plasmid R6-5 contains a locus whose product inhibits induction of sfiA and prophage lambda in a recA441 mutant at 42 degrees C and in a recA+ host after treatment with nalidixic acid. This plasmidic SOS-inhibition locus (psi) is situated on an 8.1-kilobase DNA fragment near oriT, the origin of plasmid R6-5 conjugational transfer. Loss of the Psi function, resulting from the insertion of Tn3 into psi+, greatly reduced the synthesis of two proteins, designated PsiA (Mr 24,500) and PsiB (Mr 12,500). Using host cells in which there was an inactive LexA repressor, we found that Psi function does not act by interfering with the expression of the SOS pathway. The Psi function may affect the generation of an SOS signal. We postulate that during the course of evolution, the Psi function has been selected in some conjugative plasmids so as to permit them to transfer single-stranded DNA without generating an SOS signal.

Biological Evolution↗
PubMed1985

Cold sensitivity induced by overproduction of UmuDC in Escherichia coli.

The UmuD and UmuC proteins of Escherichia coli are essential for mutagenesis by UV and most chemicals. Their mode of action is presently unknown. Strains which lack the LexA repressor [lexA(Def)] and contain a pBR322-derived plasmid carrying the umuDC operon overexpress UmuD and UmuC and become cold sensitive (growth at 42 degrees C but not at 30 degrees C). Deletion mapping showed that the umuDC locus on the plasmid is responsible for conferring cold sensitivity. The conditional lethality appeared due to a rapid and reversible inhibition of DNA synthesis at the nonpermissive temperature. Cold sensitivity was enhanced by the increase of NaCl in the medium to 1% and eliminated by 4% ethanol in the medium. Cold sensitivity was partially suppressed by the lon-100 mutation and completely suppressed by the htpR165 mutation.

Bacterial Proteins↗
PubMed1998

Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex.

The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.

Bacterial Proteins↗
PubMed1986

The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation.

A 2,695 bp chromosomal segment of Escherichia coli K12 containing the recQ gene was sequenced. Analysis of the sequence revealed an open reading frame thought to represent recQ, with a clockwise direction of transcription relative to the standard genetic map of E. coli K12 and having a coding capacity for a protein of Mr 68,350. The -10 region of the presumptive recQ promoter overlapped the putative terminator for the upstream gene pldA, and was immediately followed by a 15 bp stretch of DNA bearing a strong resemblance to the reported sequences of LexA repressor binding sites. This latter finding suggested the possibility of SOS regulation of recQ gene expression, which was substantiated by experiments with recQ-lacZ fusions.

Amino Acid Sequence↗
PubMed2001

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator.

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.

Amino Acid Sequence↗
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↗