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PubMed1988

The Escherichia coli LexA repressor-operator system works in mammalian cells.

We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells. The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40. This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein. We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene. Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter. Necessary 3' signals were from the HSV tk gene. Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor. We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.

Animals↗

PubMed1989

Autodigestion and RecA-dependent cleavage of Ind- mutant LexA proteins.

The LexA repressor of Escherichia coli undergoes a specific cleavage reaction in vivo, an event that leads to derepression of the SOS regulon and requires an activated form of RecA protein. In vitro, cleavage requires RecA at neutral pH; at alkaline pH, a spontaneous cleavage reaction termed autodigestion takes place. Both autodigestion and RecA-mediated cleavage cut the same bond, and are observed for the same set of substrates, suggesting that RecA acts indirectly to stimulate LexA self-cleavage at neutral pH, perhaps binding to LexA and acting as an allosteric effector. We previously isolated a set of lexA(Ind-) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Here, we describe the in vitro cleavage of purified mutant proteins. All of those tested were deficient in both cleavage reactions. Although most of them were equally deficient in both reactions, some were more deficient in one reaction than the other. Several mutant proteins appeared to have defects in binding to RecA. Autodigestion of all but one of the poorly cleavable mutant proteins reached a maximum rate at pH around 10, as does wild-type LexA. The exception was KR156, which changed Lys156, a residue previously implicated in the mechanism of cleavage, to Arg, another basic residue: for this protein, the rate of autodigestion increased with pH at values above 11. RecA-mediated cleavage of KR156 was 1% the wild-type rate at pH 7, but increased with increasing pH to a plateau at pH 9.5, where the rate was 40% the wild-type rate. In contrast, an essentially constant rate was observed for wild-type LexA over the pH range 6 to 11. We suggest, first, that deprotonation of Arg156 and, by inference, Lys156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage: and second, that RecA acts to reduce the pKa of Lys156, allowing efficient cleavage of wild-type repressor under physiological conditions.

Bacterial Proteins↗

PubMed1998

Reconstitution of an SOS response pathway: derepression of transcription in response to DNA breaks.

E. coli responds to DNA damage by derepressing the transcription of about 20 genes that make up the SOS pathway. Genetic analyses have shown that SOS induction in response to double-stranded DNA (dsDNA) breaks requires LexA repressor, and the RecA and RecBCD enzymes--proteins best known for their role as initiators of dsDNA break repair and homologous recombination. Here we demonstrate that purified RecA protein, RecBCD enzyme, single-stranded DNA-binding (SSB) protein, and LexA repressor respond to dsDNA breaks in vitro by derepressing transcription from an SOS promoter. Interestingly, derepression is more rapid if the DNA containing the dsDNA break has a chi recombination hot spot (5'-GCTGGTGG-3'), suggesting a novel regulatory role for one of the most overrepresented octamers in the E. coli genome.

Bacterial Proteins↗

PubMed2005

Expression of canonical SOS genes is not under LexA repression in Bdellovibrio bacteriovorus.

The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the delta-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only the lexA gene and a multiple gene cassette including dinP and dnaE homologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like the lexA gene, is DNA damage inducible in B. bacteriovorus. Conversely, genes such as recA, uvrA, ruvCAB, and ssb, which constitute the canonical core of the Proteobacteria SOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both the lexA gene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that the B. bacteriovorus LexA-binding sequence is not recognized by other delta-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the delta-Proteobacteria LexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of the Proteobacteria phylum.

Bacterial Proteins↗

PubMed1996

Mutant LexA proteins with specific defects in autodigestion.

In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure. Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule. In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein. RecA acts indirectly as a coprotease to stimulate LexA autodigestion. We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity. Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage. The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage. To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction. Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.

Amino Acid Sequence↗

PubMed2000

Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of recA.

An early event in the induction of the SOS system of Escherichia coli is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage lambda CI repressor and E. coli UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, lambda CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.

Bacterial Proteins↗

PubMed2006

A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR.

Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.

Amino Acid Sequence↗

PubMed1984

Cooperative and salt-resistant binding of lexA protein to non-operator DNA.

The interaction of the lexA repressor of E. coli with poly[d(A-T)] has been studied by circular dichroism. The binding induces an about 2-fold increase of the circular dichroism intensity at 263 nm, pointing out a conformational change of the nucleic acid. The observed spectral changes are very similar to those observed for the binding of the lac repressor to poly[d(A-T)] and natural DNA. At elevated ionic strength the binding isotherms do show a pronounced sigmoidal shape indicating a cooperative mode of binding.

Bacterial Proteins↗

PubMed2005

Inhibition of mutation and combating the evolution of antibiotic resistance.

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.

Animals↗

PubMed1983

The muc genes of pKM101 are induced by DNA damage.

A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.

Bacterial Proteins↗

PubMed1991

Interaction of a regulatory protein with a DNA target containing two overlapping binding sites.

The LexA repressor from Escherichia coli regulates the transcription of about 20 different genes upon binding to single or multiple operators. In this work we study the interaction of LexA with the control region of the caa gene (coding for the bacterial toxin colicin A) that contains two operators (O1 and O2) which overlap by at least 2 base pairs relevant for sequence specific DNA recognition. This arrangement raises the question of how the LexA molecules which bind to the central overlapping part of the two operators avoid steric clashes and further, of whether the interaction of LexA with the two operators is cooperative or not. To address these questions we have constructed two mutant operators (O1+O2- and O1-O2+) for which the two most strongly conserved base pairs in each of the external operator half-sites have been mutated. Using methylation interference with the complex formation of LexA with the wild-type and these two mutant operators we could show: 1) that the two mutant operators behave symmetrically in that the methylation of one crucial guanine base in both operator half-sites interferes strongly with complex formation, 2) but that in the wild-type operator (containing four functional operator half-sites) only the two external half-operators give rise to interference if this crucial guanine base is methylated, whereas methylation of the two equivalent guanine bases within the two central (overlapping) operator half-sites does not lead to interference with the formation of a complex where both operators are occupied simultaneously. These data suggest that the centrally bound LexA molecules adopt a somewhat different binding mode than those bound to the external half-operators in order to avoid steric clashes and/or to optimize protein-protein contacts which are likely to be at the origin of the binding cooperativity that we could demonstrate by quantitative DNase I footprinting and gel retardation experiments. While the methylation interference experiments revealed a non-equivalence for the binding of externally and centrally bound LexA molecules, both methylation protection and hydroxyl radical footprinting were unable to reveal this difference, suggesting that the difference between the two binding modes should be fairly subtle.

Bacterial Proteins↗

PubMed1993

Inhibition of DNA replication factor RPA by p53.

The tumour suppressor p53 specifically interferes with the onset of S phase. The mechanism of the growth suppression action of the protein is unclear, though recent evidence points to transcriptional activation and repression functions of the protein. A competing hypothesis suggests that p53 interacts with the DNA replication apparatus and directly interferes with DNA replication. The major evidence for this hypothesis is that p53 interacts with the simian virus 40 (SV40)-encoded protein T antigen and interferes with the ability of T antigen to unwind the SV40 origin of DNA replication, and recruit DNA polymerase alpha to the replication initiation complex. Here we report that p53 physically interacts with and inhibits the function of a cellular DNA replication factor, the single-stranded DNA-binding protein complex RPA.

Antigens, Polyomavirus Transforming↗

PubMed1999

Steroid receptor coactivator-1 and its family members differentially regulate transactivation by the tumor suppressor protein p53.

The tumor suppressor protein p53 exerts its cell cycle-regulatory effects through its ability to function as a sequence-specific DNA-binding transcription factor. Herein, we show that p53 physically interacts with specific subregions of steroid receptor coactivator-1 (SRC-1) and its family members, p/CIP (p300/CBP interacting protein), xSRC-3, and AIB1 (amplified in breast cancer), originally isolated as transcription coactivators of nuclear receptors, as demonstrated by the yeast and mammalian two-hybrid tests as well as glutathione S-transferase pull-down assays. Interestingly, cotransfection of HeLa cells with SRC-1- or p/CIP expression vector potentiated the p53-mediated transactivation, whereas AIB1 and xSRC-3 were repressive. All of these SRC-1 members, however, similarly stimulated transactivation mediated by nuclear receptors and AP-1, as previously described. These results suggest that SRC-1 and its family members may differentially modulate the p53 transactivation in vivo.

Acetyltransferases↗

PubMed1988

The carboxy-terminal domain of the LexA repressor oligomerises essentially as the entire protein.

The ability of the isolated carboxy-terminal domain of the LexA repressor of Escherichia coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85-202, is readily purified after self-cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy-terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non-linear least squares fitting of the experimental concentration distribution. A dimer association constant of K2 = 3 X 10(4) M-1 and a tetramer association constant of K4 = 2 X 10(4) M-1 have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812-2818] gave values of K2 = 2.1 X 10(4) M-1 and K4 = 7.7 X 10(4) M-1. Within experimental error the dimer formation constant of the carboxy-terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K4 is higher than that in K2. The overall conclusion is that the two structural domains of LexA have also well-defined functional roles: the amino-terminal domain interacts with DNA and the carboxy-terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.

Bacterial Proteins↗

PubMed1989

Mutation of the promoter and LexA binding sites of cea, the gene encoding colicin E1.

Three mutations were introduced into the cea promoter using oligonucleotide directed mutagenesis. The resulting mutant promoter has the Escherichia coli consensus sequences at its - 35 and - 10 positions, separated by the optimal spacing. In addition, a plasmid with a mutation in one of the two LexA repressor binding sites in the cea regulatory region was isolated that decreases homology with the consensus LexA binding site. The effects of these mutations on cea expression were studied in cea-lacZ protein fusions. The promoter-up mutant, when present in a multicopy plasmid, showed a shorter induction lag when compared to the wild-type cea gene, and there was less of an effect of the catabolite repression system on cea expression. However, when present in a single copy in the bacterial chromosome, catabolite repression and an induction delay were observed, despite the increased strength of the promoter. The operator mutant showed a slightly higher basal level of expression, but was still repressible. Induction occurred with a shortened lag period, but the effects were not as great as with the promoter mutant. These results support the idea that tight repression by LexA contributes to the delay in cea induction.

Bacterial Proteins↗

PubMed1992

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 bp in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 bp in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84--Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.

Amino Acid Sequence↗

PubMed1998

Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae.

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.

Bacterial Proteins↗

PubMed1994

The protein HU can displace the LexA repressor from its DNA-binding sites.

The major bacterial histone-like protein HU is a small, basic, dimeric protein composed of two closely related subunits. HU is involved in several processes in the bacterial cell such as the initiation of replication, transposition, gene inversion and cell division. It has been suggested that HU could introduce structural changes to the DNA which would facilitate or inhibit the binding of regulatory proteins to their specific sites. In this study we investigated the effect of HU on the binding of LexA protein, the regulator of SOS functions, to three of its specific binding sites. We show that HU can displace LexA from its binding sites on the operators of the lexA, recA and sfiA genes. The lexA operator was the most sensitive while the higher affinity sfiA operator was the least sensitive. Since HU, like its homologue IHF, probably binds DNA in the minor groove we tested the effect of distamycin, a drug which binds to the minor groove, on LexA binding. Like HU, this drug disrupted LexA-operator complexes. These results suggest that distortion of the minor groove of the lexA operators excludes the binding of the repressor to the major groove.

Bacterial Proteins↗