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Results for “LexA Repressor Protein”

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PubMed1999

A two-hybrid dual bait system to discriminate specificity of protein interactions.

Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.

Bacterial Proteins↗

PubMed1998

A LexA mutant repressor with a relaxed inter-domain linker.

The LexA protein is part of a large family of prokaryotic transcriptional repressors that contain an amino-terminal DNA binding domain and a carboxy-terminal dimerization domain. These domains are separated by a linker or hinge region, which is generally considered to be rather flexible and unconstrained. So far, no structure of any of the full-length repressors is available. Here we show that a mutant LexA repressor harboring several point mutations in the hinge region gets sensitive to trypsin and Glu-C cleavage over a segment of at least 20 amino acids, whereas the LexA wild-type hinge region is resistant to these proteases. These data are not compatible with the hypothesis of an fully flexible and/or unstructured inter-domain linker and suggest that the LexA hinge region is, in fact, constrained by contacts with the carboxy-terminal domain and/or a fairly stable local structure of the linker region.

Amino Acid Sequence↗

PubMed2002

Connection between gene dosage and protein stability revealed by a high-yield production of recombinant proteins in an E. coli LexA1(Ind-) background.

Bacterial production of a plasmid-encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA- LexA+ and RecA+ LexA1(Ind-) backgrounds. In a LexA1(Ind-) context, we have observed lesser toxicity and higher productivity than in the wild-type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression. Also, a negative effect of the incubation temperature on the growth of producing cells was also detected. By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein. Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition. Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth.

Bacterial Proteins↗

PubMed1984

Regulation and SOS induction of division inhibition in Escherichia coli K12.

When Escherichia coli is subjected to treatments that damage DNA or perturb DNA replication considerable cell filamentation occurs. It has been postulated that this phenomenon is associated with the presence of a division inhibitor induced coordinately with the SOS functions. The role of this induction would be to delay septation during DNA repair to prevent the formation of DNAless cells. In this communication, we present evidence for such a division inhibitor based on the properties of a division mutant which is hyperactive in the septation delay. Cells of this mutant filament extensively after a nutritional shift-up, have drastically reduced colony-forming abilities on a rich medium but not on a minimal medium following treatment with ultraviolet radiation and, are deficient in the lysogenization of phage lambda; phenotypes which are characteristic of but expressed to a much lower extent in another type of division mutant called Ion. Cells harboring the division mutation plus either one of the lexA mutant alleles, spr-51 or tsl-1, are filamentous suggesting that they are permanently derepressed for division inhibition. These results are in agreement with models that assign the regulation of cell division to a division inhibitor which is regulated by the lexA repressor protein.

Bacteriophage lambda↗

PubMed1985

Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon.

We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam-). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of beta-galactosidase in dam- strains than in isogenic dam+ strains. The attempted construction of dam- strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam- strain viability.

DNA Repair↗

PubMed1991

Self-cleaving proteases.

Research on the activity of self-cleaving proteases in bacterial, mammalian and virus-infected cells is reviewed, with an emphasis on the diversity of regulatory systems controlled by protein processing. Each of these three groups will be considered in turn by focusing on the following systems: the Rec A-dependent intramolecular cleavage of the Escherichia coli SOS response protein, LexA; the intramolecular activation of the mammalian aspartic acid protease, pepsinogen; and the autocatalytic cleavage of polyproteins synthesized by picornaviruses.

Bacterial Proteins↗

PubMed1998

Transactivation potential of the C-terminus of human Nm23-H1.

The transactivation potential of Nm23-H1, a homolog of c-myc transcription factor Nm23-H2/PuF was assessed in yeast as a fusion protein with the DNA binding domains (DBDs) of GAL4 and LexA. The C-terminal half of Nm23-H1 exhibited strong transactivation of the reporter genes, LacZ and Leu2 carrying GAL4 and LexA upstream activating sequences (UASs), whereas the full-length Nm23-H1 and its N-terminal did not. Similar results were also obtained with Nm23-H2/PuF transactivating the reporter genes only by the C-terminus fused to GAL4 and LexA DBDs. Hence, our results suggested a possible regulatory role of the N-termini of Nm23 isotypes upon transactivation.

Bacterial Proteins↗

PubMed2004

The nuclear protein p34SEI-1 regulates the kinase activity of cyclin-dependent kinase 4 in a concentration-dependent manner.

Previous studies have shown that p34(SEI-1), also known as TRIP-Br1, is involved in cell cycle regulations by interacting with a number of important proteins including CDK4. However, the detailed mechanism and structural basis of the interaction remains to be determined. We report the use of in vitro studies to address these problems. First, it was shown that p34(SEI-1) binds to CDK4 directly, and the binding does not compete directly with p16. In the presence of p16, a quaternary complex is formed between p34(SEI-1), CDK4, cyclin D2, and p16. Second, it was found that p34(SEI-1) activates the kinase activity of CDK4 at lower concentrations (reaching the maximum at 500 nM) but inhibits the same activity at higher concentrations, implying that p34(SEI-1)-mediated CDK4 activation is dose-dependent. Again, the effects of p34(SEI-1) and p16 are independent of each other. Third, it was shown that p34(SEI-1) possesses a LexA-mediated transactivation activity. Finally, a set of truncation mutants were used to dissect the structural elements responsible for the different functions of p34(SEI-1). The results indicate that the fragment 30-160 can bind, activate, and inhibit CDK4; the fragment 30-132 can bind and activate but does not inhibit CDK4, while the fragment 30-88 cannot bind, activate, or inhibit but retains the LexA-mediated transactivation activity.

Amino Acid Motifs↗

PubMed1989

Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744.

Escherichia coli that carry a recombinant plasmid bearing the Vibrio fischeri lux regulon express luminescence that mimics the luminescence of V. fischeri. The lux regulon consists of two divergently transcribed operons, the rightward operon (luxICDABE genes) and the leftward operon (luxR gene). The luxR and luxI genes and the control region separating the two operons supply the primary regulatory control over the lux regulon; the regulatory mechanisms result in a dramatic increase in the rate of luciferase synthesis after induction, apparently due to a unique autoregulatory positive feedback mechanism, and in an enormous difference (greater than 10(4] in levels of luminescence in cells before and after induction. The generally accepted model of primary regulation of bioluminescence in V. fischeri involves the interaction of the product of the luxR gene and N-(3-oxohexanoyl)homoserine lactone, the autoinducer produced by the enzyme encoded by luxI, the first gene of the rightward operon, with an operator sequence within the control region to stimulate transcription of the rightward operon in a positive feedback loop. We have used deletion mapping of a transcription reporter vector to determine the approximate location of the operator. By site-directed mutagenesis of the presumed operator, we have demonstrated that the 20-base-pair inverted repeat ACCTGTAGGA/TCGTA CAGGT (where the vertical line is the center of symmetry), which bears striking similarity to the recognition sequence for the pleiotropic repressor protein LexA, is the operator of the lux regulon. We also found that deletion of sequences upstream of the palindrome leads to increased transcription from the rightward promoter (PR), indicative of a cis-acting element that represses transcription in the absence of the LuxR-autoinducer complex. Modifications of the palindrome that eliminate stimulation by LuxR-autoinducer of transcription from PR have no effect on repression by the cis-acting mechanism(s), suggesting that the palindrome is not necessary for repression of the rightward operon. Thus, it appears that the large increase in transcription upon induction of the lux regulon is the result of at least two independent mechanisms, one positive and the other negative.

Amino Acid Sequence↗

PubMed2002

Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage.

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind(-)) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.

Bacterial Proteins↗

PubMed1996

Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.

Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.

Bacterial Proteins↗

PubMed2004

What is the structure of the RecA-DNA filament?

The bacterial RecA protein has been a model system for understanding how a protein can catalyze homologous genetic recombination. RecA-like proteins have now been characterized from many organisms, from bacteriophage to humans. Some of the RecA-like proteins, including human RAD51, appear to function as helical filaments formed on DNA. However, we currently have high resolution structures of inactive forms of the protein, and low resolution structures of the active complexes formed by RecA-like proteins on DNA in the presence of ATP or ATP analogs. Within a crystal of the E. coli RecA protein, a helical polymer exists, and it has been widely assumed that this polymer is quite similar to the active helical filament formed on DNA. Recent developments have suggested that this may not be the case.

Adenosine Triphosphatases↗

PubMed1980

Cleavage of the Escherichia coli lexA protein by the recA protease.

The recA and lexA proteins of EScherichia coli are involved in a complex regulatory circuit that allows the expression of a diverse set of functions after DNA damage or inhibition of DNA replication. Exponentially growing cells contain a low level of recA protein, and genetic evidence suggests that lexA protein is involved in its regulation, perhaps as a simple repressor. Recent models for recA derepression after DNA damage have suggested that an early event in this process is the proteolytic cleavage of lexA protein, leading to high-level expression of recA. We present several lines of evidence that the specific protease activity of the recA protein, previously described with the lambda repressor as substrate, is capable of cleaving the wild-type lexA+ protein. First, lexA protein can be cleaved in vitro under the same conditions as prevously described for lambda repressor cleavage in a reaction requring both recA protease and ATP or an analogue, adenosine 5'-[lambda-thio]-triphosphate. Second, lexA protein can be observed in vivo as a physical entity after infection with lambda lexA+ transducing phage of host strains containing ittle or no active protease, but not in strains containing high levels of active protease. Finally, infection of host cells containing active protease with a lambda lexA+ transducing phage does not lead to repression of recA, but does so in cells lacking active protease. In all of these conditions the mutant lexA3 protein is largely resistant to inactivation or cleavage; this resistance can explain the dominant phenotype of lexA3 over lexA+. We discuss models for recA derepression and re-establishment of repression which propose that modulation of the protease activity of recA protein regulates both of these transitions.

Adenosine Triphosphatases↗

PubMed1985

Deletions within a hinge region of a specific DNA-binding protein.

Many proteins are organized as a set of compact functional domains connected by flexible, exposed segments of the polypeptide chain. To study one of these connector regions, we isolated a series of functional in-frame deletions in the central portion of a specific DNA-binding protein, the LexA repressor of Escherichia coli. These mutant proteins fell into two main classes: those with small deletions of two to eight amino acids functioned as repressor about as well as did wild type, while those with large deletions of 17-22 amino acids functioned well only at considerably higher concentrations. The mutant proteins were resistant to the specific cleavage reaction that triggers the SOS response. These data suggest that the conformation of the hinge region in LexA protein is important for cleavage. By contrast, the hinge plays a topological role in repressor function, connecting the two functional halves of the protein; in the SOS response, this function of the hinge is inactivated by cleavage, leading to inactivation of the repressor.

Amino Acid Sequence↗

PubMed1986

In vitro study of the interaction of the LexA repressor and the UvrC protein with a uvrC regulatory region.

The in vitro interaction of the LexA repressor with a regulatory region of the uvrC gene has been studied by polyacrylamide gel electrophoresis. Although the uvrC promoter region shows some homology with the canonic LexA binding site, no specific binding of the repressor to this DNA sequence could be observed, but only a cooperative nonspecific binding. By the same technique we show that the UvrC protein does not bind specifically to this regulatory DNA sequence either, although the protein is able to bind nonspecifically and cooperatively to the double-stranded DNA fragment.

Bacterial Proteins↗

PubMed2002

A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa.

Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA-recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN(6)TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN(6)TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA-recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.

Bacterial Proteins↗

PubMed1991

MAT alpha 1 can mediate gene activation by a-mating factor.

In the yeast Saccharomyces cerevisiae, expression of alpha-specific genes is governed by the MAT alpha 1 and MCM1 gene products. MAT alpha 1 and MCM1 bind cooperatively to PQ elements upstream of alpha-specific genes. The PQ element not only directs alpha-specific expression but can also direct gene induction in response to treatment with a-mating pheromone. We have used gene fusions to investigate whether induction conferred by the PQ box is mediated through either MAT alpha 1 or MCM1, or a combination of both. When MCM1 is fused to the DNA-binding domain of the bacterial repressor LexA, this fusion protein is capable of trans-activating a lacZ reporter gene driven by a LexA operator. However, the transcriptional activity of the MCM1-LexA fusion is not further enhanced by treatment of cells with a-factor. A MAT alpha 1-LexA fusion protein is also capable of trans-activation through a LexA operator. Moreover, the activity of the MAT alpha 1-LexA fusion protein can be further induced by treatment with a-factor. When progressive deletions are made from the amino terminus of MAT alpha 1 in the fusion protein, the basal level of trans-activation progressively decreases, but the inducibility of the fusion protein increases. MAT alpha 1-LexA fusion proteins, which have greater than or equal to 57 amino acids deleted from the amino terminus of MAT alpha 1 are not capable of trans-activation. In addition, the activity of the MAT alpha 1-LexA fusion protein is dependent on the functions of the STE7, STE11, and STE12 genes that encode components of the pheromone response pathway.

Amino Acid Sequence↗

PubMed1991

The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad.

General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.

Bacterial Proteins↗