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PubMed1994

Membrane permeability and sensitivity to lethal heat are affected by lexA and recA mutations in Escherichia coli K12.

Membrane permeability was evaluated in several SOS-deficient strains. Great heat sensitivity was observed in all the lexA (Ind-) strains, which was associated to an increase in membrane permeability (up to 120% increase above the wild-type control), as assayed by the crystal violet (CV) growth inhibition. After irradiation with a single UV dose (75 J.m-2 delivered to wild-type and 2 J.m-2 to the lexA3 strain), survival was followed by plating cells in both nutrient and membrane permeability-selective (nutrient + CV) media and a great lethality due to CV was observed in a lexA mutant, which appeared to be about 100 times more sensitive to CV compared to its wild-type parent strain. The decreased membrane integrity found in the lexA-deficient strains suggests that LexA protein and/or LexA-repressed genes may interact with the bacterial membrane, which could be the location of SOS events.

Bacterial Proteins↗
PubMed2004

Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation.

The cyanobacterial genes lexA, recA and ruvB were analysed in Synechocystis PCC6803, which is shown here to be more radiation resistant than the other unicellular model strain Synechococcus PCC7942. We found that cyanobacteria do not have an Escherichia coli-type SOS regulon. The Synechocystis lexA and recA promoters were found to be strong and UV insensitive, unlike the ruvB promoter, which is weak and UV-C inducible. Yet, lexA and recA are regulated by UV-C, but the control is negative and occurs at the post-transcriptional level. Two novel conserved elements were characterized in the lexA promoter: (i) an unusually long crucial box 5'-TAAAATTTTGTATCTTTT-3' (-64, -47); and (ii) a negatively acting motif 5'-TAT GAT-3' (-42, -37). These elements were not found in the recA promoter, which appeared to be unusually simple in harbouring only a single crucial element (i.e. the canonical -10 box). RuvB, operating in recombination-dependent cellular processes, was found to be dispensable to cell growth, whereas LexA and RecA appeared to be critical to cell viability. Using DNA microarrays, we have identified 57 genes with expression that is altered, at least twofold, in response to LexA depletion. None of these genes is predicted to operate in DNA metabolism, arguing against the involvement of LexA in the regulation of DNA repair. Instead, most of the LexA-responsive genes were known to be involved in carbon assimilation or controlled by carbon availability. Consistently, the growth of the LexA-depleted strain was found to be strongly dependent on the availability of inorganic carbon.

Bacterial Proteins↗
PubMed1993

Analysis of the DNA damage-mediated induction of Pseudomonas putida and Pseudomonas aeruginosa lexA genes.

A fusion between the lexA gene of Pseudomonas aeruginosa and Pseudomonas putida and the lacZ gene was constructed in vitro and cloned in a mini-Tn5 transposon derivative to obtain chromosomal insertions which enable to quantitatively examine their transcriptional regulation in both Pseudomonas and E. coli. Analysis of DNA damage-mediated induction of these lexA-lacZ fusions showed that expression of P. putida and P. aeruginosa lexA genes was always higher and earlier than the expression of the lexA gene of E. coli. Furthermore, and in contrast to the lexA gene fusion of E. coli, the rates and extent of the induction of lexA gene fusion of P. putida and P. aeruginosa were largely independent of the UV doses applied. The behaviour of the lexA-lacZ fusions of two Pseudomonas species was the same regardless of whether they were inserted into their own chromosome or into E. coli.

Bacterial Proteins↗
PubMed1992

Interaction of LexA repressor with the asymmetric dinG operator and complete nucleotide sequence of the gene.

The dinG gene was originally isolated during a search for Escherichia coli promoters which are components of the SOS regulon. The regulatory region of this gene contains a potential binding site for LexA repressor which is quite different from other known sites. All previously described chromosomal LexA operators are imperfect palindromes containing the sequence CTG(N10)CAG. The noncanonical dinG sequence breaks the symmetry and takes the form TTG(N10)CAG. In the present study, a search for mutations within dinGop::galK fusion plasmids which render transcription independent of intracellular levels of LexA has yielded mutations only within this 16-bp sequence. Electrophoretic mobility shift assays performed with purified mutant and wild-type operator fragments revealed that the affinity of LexA for each of the mutant sites is greatly reduced compared with that of the wild type. One of the mutants contained an alteration in the putative promoter of dinG which increased the similarity of the -35 region to the consensus sequence (TTGGCT----TTGACT); the apparent promoter activity of this construct was subsequently found to be approximately eight times higher than that of the wild type in vivo. Additional experiments have established the complete nucleotide sequence of the dinG gene. A long open reading frame located immediately downstream of the asymmetric operator segment which could potentially encode a 72.9-kDa DinG protein was identified.

Amino Acid Sequence↗
PubMed2001

Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri.

The role of the LexA protein and, specifically, its effect on recA expression were analyzed in Xanthomonas campestris pathovar citri (X.c. pv. citri). Overexpression of LexA from X.c. pv. citri, in the plant pathogen, as well as in Escherichia coli, results in increased sensitivity to the DNA-damaging agents mitomycin C and ultraviolet radiation, indicating that the recombinant X.c. pv. citri LexA protein is functional in a different bacterial species. Immunoblot analysis revealed that the overexpressed LexA protein functioned as a repressor of recA expression in X.c. pv. citri, and that the mitomycin C-induced increase in the abundance of RecA was accompanied by specific proteolysis of LexA that required RecA. Although the LexA protein from X.c. pv. citri also blocked the expression of recA in E. coli, the E. coli RecA protein was not able to support the autocatalytic cleavage of LexA from the plant pathogen. The transcription start site of the X.c. pv. citri lexA gene was identified, and the region upstream of this gene was shown to confer responsiveness to mitomycin C on a luciferase reporter gene construct. Electrophoretic mobility-shift assays demonstrated that X.c. pv. citri LexA interacts with the promoter region of X.c. pv. citri lexA, as well as with those of the recA genes of X.c. pv. citri and E. coli. These results indicate that LexA functions as a repressor of gene expression in X.c. pv. citri just as it does in E. coli.

Bacterial Proteins↗
PubMed2005

Binding of the Bacillus subtilis LexA protein to the SOS operator.

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5'-CGAACN(4)GTTCG-3'. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5' end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5'-CGAACATATGTTCG-3'. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.

Bacillus subtilis↗
PubMed2006

Dispersal and regulation of an adaptive mutagenesis cassette in the bacteria domain.

Recently, a multiple gene cassette with mutagenic translation synthesis activity was identified and shown to be under LexA regulation in several proteobacteria species. In this work, we have traced down instances of this multiple gene cassette across the bacteria domain. Phylogenetic analyses show that this cassette has undergone several reorganizations since its inception in the actinobacteria, and that it has dispersed across the bacterial domain through a combination of vertical inheritance, lateral gene transfer and duplication. In addition, our analyses show that LexA regulation of this multiple gene cassette is persistent in all the phyla in which it has been detected, and suggest that this regulation is prompted by the combined activity of two of its constituent genes: a polymerase V homolog and an alpha subunit of the DNA polymerase III.

Adaptation, Physiological↗
PubMed1997

Characterization of the fatty acid-responsive transcription factor FadR. Biochemical and genetic analyses of the native conformation and functional domains.

In Escherichia coli, fatty acid synthesis and degradation are coordinately controlled at the level of transcription by FadR. FadR represses transcription of at least eight genes required for fatty acid transport and beta-oxidation and activates transcription of at least two genes required for unsaturated fatty acid biosynthesis and the gene encoding the transcriptional regulator of the aceBAK operon encoding the glyoxylate shunt enzymes, IclR. FadR-dependent DNA binding and transcriptional activation is prevented by long chain fatty acyl-CoA. In the present work, we provide physical and genetic evidence that FadR exists as a homodimer in solution and in vivo. Native polyacrylamide gel electrophoresis and glycerol gradient ultracentrifugation of the purified protein show that native FadR was a homodimer in solution with an apparent molecular mass of 53.5 and 57.8 kDa, respectively. Dominant negative mutations in fadR were generated by random and site-directed mutagenesis. Each mutation mapped to the amino terminus of the protein (residues 1-66) and resulted in a decrease in DNA binding in vitro. In an effort to separate domains of FadR required for DNA binding, dimerization, and ligand binding, chimeric protein fusions between the DNA binding domain of LexA and different regions of FadR were constructed. One fusion, LexA1-87-FadR102-239, was able to repress the LexA reporter sulA-lacZ, and beta-galactosidase activities were derepressed by fatty acids, suggesting that the fusion protein had determinants both for dimerization and ligand binding. These studies support the conclusion that native FadR exists as a stable homo-dimer in solution and that determinants for DNA binding and acyl-CoA binding are found within the amino terminus and carboxyl terminus, respectively.

Alleles↗
PubMed1993

Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction.

The recA432 mutant allele was isolated (T. Kato and Y. Shinoura, Mol. Gen. Genet. 156:121-131, 1977) by virtue of its defect in cellular mutagenesis (Mut-) and its hypersensitivity to damage by UV irradiation (UVs), which were phenotypes expected for a recA mutant. However, we found that in a different genetic background (lexA51 sulA211 uvrB+), recA432 mutants expressed certain mutant phenotypes but not the Mut- and UVs phenotypes (D.G. Ennis, N. Ossanna, and D.W. Mount, J. Bacteriol. 171:2533-2541, 1989). We present several lines of evidence that these differences resulted from the sulA genotype of the cell and that the apparent UVs and Mut- phenotypes of the sulA+ derivatives resulted from lethal filamentation of induced cells because of persistent derepression of sulA. First, transduction of sulA(Def) mutations into the recA432 strains restored cellular mutagenesis and resistance to UV. Second, recA432 sulA+ strains underwent filamentous death following SOS-inducing treatments. Third, cleavage of LexA repressor in a recA432 strain continued at a rapid rate long after UV induction, at a time when cleavage of the repressor in the recA+ parental strain had substantially declined. Fourth, we confirmed that a single mutation (recA432) conferring both the UVs and Mut- phenotypes mapped to the recA gene. These findings indicate that the RecA432 mutant protein is defective in making the transition back to the deactivated state following SOS induction; thus, the SOS-induced state of recA432 mutants is prolonged and can account for an excess of SulA protein, leading to filamentation. These results are discussed in the context of molecular models for RecA activation for LexA and UmuD cleavage and their roles in the control of mutagenesis and cell division in the SOS response.

Bacterial Proteins↗
PubMed2005

Genetic composition of the Bacillus subtilis SOS system.

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

5' Flanking Region↗
PubMed2001

Molecular characterization and expression of the recX gene of Xanthomonas campestris pv. citri.

Two genes important in DNA repair, recA and lexA, were recently identified in Xanthomonas campestris pathovar citri (X.c. pv. citri). An open reading frame located immediately downstream of lexA and recA has now been isolated from this pathovar and characterized. This 486-bp open reading frame encodes a protein of 162 amino acids and shares substantial sequence similarity with recX of other bacterial species. The X.c. pv. citri RecX protein was overexpressed in Escherichia coli and purified; SDS-polyacrylamide gel electrophoresis revealed a molecular size of 18 kDa for the purified protein. Whereas Northern blot analysis failed to detect recX mRNA in X.c. pv. citri, recX transcripts were detected in this pathovar by reverse transcription and polymerase chain reaction analysis. The increased abundance of recX transcript in X.c. pv. citri revealed that the recX promoter was activated by exposure of cells to DNA-damaging agents. Southern blot and polymerase chain reaction analyses revealed the presence of a recX-related gene in all nine additional X. campestris pathovars tested. The genetic arrangement of lexA-recA-recX was apparent in X. campestris and each of the three genes transcribed from their own promoters.

Amino Acid Sequence↗
PubMed1993

Functional analysis of a growth factor-responsive transcription factor complex.

Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory factor, Elk-1. We constructed altered-binding specificity derivatives of SRF and Elk-1 that form a ternary complex at a mutated, inactive SRE; like Elk-1, the Elk-1 variant only binds its target as part of a ternary complex with SRF. Simultaneous expression of these SRF and Elk-1 derivatives restores serum-regulated activity to the mutated SRE in transfected cells. Efficient transcriptional activation is dependent on the regulated phosphorylation of Elk-1 C-terminal MAP kinase sites and requires the C-terminal sequences of SRF as well as SRF sequences that mediate ternary complex formation. These experiments provide direct evidence that SRF and Elk-1 functionally cooperate in the ternary complex at the SRE to regulate transcription.

3T3 Cells↗
PubMed1999

Intersubunit proximity of residues in the RecA protein as shown by engineered disulfide cross-links.

Mutational studies of regions that make up the oligomeric interface within the RecA protein filament structure have shown that F217 is an important determinant of RecA function and oligomer stability. All substitutions, other than Tyr and Cys, completely inhibit RecA activities and exhibit a substantial decrease in protein filament stability [Skiba, M. C., and Knight, K. L. (1994) J. Biol. Chem. 269, 3823-3828; Logan, K. M., et al. (1997) J. Mol. Biol. 266, 306-316]. Although the RecA crystal structure exhibits no obvious constraints that explain this mutational stringency, the structure does reveal a hydrophobic pocket in the neighboring monomer that may accommodate the F217 side chain. Together with the F217C mutation, we have introduced a series of Cys substitutions within the interacting surface on the neighboring monomer and have tested for disulfide formation under various conditions, e.g., with or without ATP and ssDNA. We show that the location of F217 in the crystal structure is in general agreement with its position in the catalytically active RecA-ATP-DNA complex. Functional studies with the mutant proteins support the idea that ATP-induced movement of the wild-type F217 side chain toward this hydrophobic pocket is important in mediating allosteric changes in the RecA protein structure.

Bacterial Proteins↗
PubMed2003

Controlling integration specificity of a yeast retrotransposon.

Retrotransposons and retroviruses integrate nonrandomly into eukaryotic genomes. For the yeast retrotransposon Ty5, integration preferentially occurs within domains of heterochromatin. Targeting to these locations is determined by interactions between an amino acid sequence motif at the C terminus of Ty5 integrase (IN) called the targeting domain, and the heterochromatin protein Sir4p. Here we show that new Ty5 integration hot spots are created when Sir4p is tethered to ectopic DNA sites. Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either IN or Sir4p that prevent their interaction. Ty5 target specificity can be altered by replacing the IN-targeting domain with other peptide motifs that interact with known protein partners. Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners are tethered. These findings define a mechanism by which retrotransposons shape their host genomes and suggest ways in which retroviral integration can be controlled.

Bacterial Proteins↗
PubMed1996

A three-hybrid system for detecting small ligand-protein receptor interactions.

Small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine. We report herein a genetic system, named the yeast three-hybrid system, for detecting ligand-receptor interactions in vivo. This system is adapted from the yeast two-hybrid system with which a third synthetic hybrid ligand is combined. The feasibility of this system was demonstrated using as the hybrid ligand a heterodimer of covalently linked dexamethasone and FK506. Yeast expressing fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and FKBP12 fused to a transcriptional activation domain activated reporter genes when plated on medium containing the dexamethasone-FK506 heterodimer. The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506. Using this system, we screened a Jurkat cDNA library fused to the transcriptional activation domain in yeast expressing the hormone binding domain of rat glucocorticoid receptor-LexA DNA binding domain fusion protein in the presence of dexamethasone-FK506 heterodimer. We isolated overlapping clones of human FKBP12. These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors.

Animals↗
PubMed1995

Evidence for a differential interaction of SHC and the insulin receptor substrate-1 (IRS-1) with the insulin-like growth factor-I (IGF-I) receptor in the yeast two-hybrid system.

Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr-1131, -1135, and -1136) of the IGF-IR, and (iv) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Multimerization of Hsp42p, a novel heat shock protein of Saccharomyces cerevisiae, is dependent on a conserved carboxyl-terminal sequence.

Rap1p is a transcriptional regulator of Saccharomyces cerevisiae, which plays roles in both transcriptional activation and silencing. To identify proteins involved in Rap1p-dependent regulation of transcription, we used the two-hybrid system to screen for Rap1p-interacting proteins. Two of the clones isolated from this screen encode a truncated protein with homology to small heat shock proteins (HSPs). Here we present an analysis of this novel S. cerevisiae HSP, which we name Hsp42p. Expression of HSP42 is regulated by a range of stress conditions similar to S. cerevisiae HSP26, with which Hsp42p shares most homology. However, HSP42 expression is more sensitive to increased salt concentration and to starvation and, in contrast to HSP26 is expressed in unstressed cells. Hsp42p interacts with itself in the two-hybrid assay. This interaction is dependent on a hydrophobic region which is conserved among small HSPs. Using bacterially expressed Hsp42p fusion proteins. we demonstrate that this is a direct interaction. Fractionation of yeast protein extracts by size demonstrates that all of the Hsp42p in these extracts is present in complexes with a molecular mass of greater than 200 kDa, suggesting that Hsp42p exists in high molecular mass complexes.

Amino Acid Sequence↗
PubMed2001

Elk-1, C/EBPalpha, and Pit-1 confer an insulin-responsive phenotype on prolactin promoter expression in Chinese hamster ovary cells and define the factors required for insulin-increased transcription.

The transcription factor(s) that mediate insulin-increased gene transcription are not well defined. These studies use phenotypic conversion of Rat2 and Chinese hamster ovary (CHO) cells with transcription factors to identify components required for regulation of prolactin promoter activity and its control by insulin. The pituitary-derived GH4 cells contain all of the transcription factors required for insulin-increased prolactin-chloramphenicol acetyltransferase (CAT) expression while HeLa cells require only Pit-1, a pituitary-specific factor. However, Rat2 and CHO cells require additional factors. We had determined previously that the transcription factor that mediates insulin-increased prolactin gene expression was likely an Ets-related protein. Elk-1 and Sap-1 were the only Ets-related transcription factors tested as chimeras with LexA DNA-binding domain that were able to mediate insulin-increased expression of a LexA-CAT reporter plasmid. Elk-1 and Sap-1 are expressed in GH4 and HeLa cells but Rat2 and CHO cells express Sap-1, but not Elk-1. Expression of Elk-1 made Rat2 cells (but not CHO cells) insulin responsive. C/EBPalpha also binds to the prolactin promoter at a sequence overlapping the binding site for Elk-1. Expression of both C/EBPalpha and Pit-1 in CHO cells is required for high basal transcription of prolactin-CAT. Expression of Elk-1 converts CHO cells into a phenotype in which prolactin gene expression is increased by insulin treatment. Finally, antisense mediated reduction of Elk-1 in GH4 cells decreased insulin-increased prolactin gene expression and confirmed the requirement for Elk-1 for insulin-increased prolactin gene expression. Thus, both C/EBPalpha and Pit-1 were required for high basal transcription while insulin sensitivity required Elk-1.

3T3 Cells↗