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PubMed1992

Fused protein domains inhibit DNA binding by LexA.

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12.

The specificity of LexA protein binding was investigated by quantifying the repressibility of several mutant recA and lexA operator-promoter regions fused to the Escherichia coli galactokinase (galK) gene. The results of this analysis indicate that two sets of four nucleotides, one set at each end of the operator (terminal-nucleotide contacts), are most critical for repressor binding. In addition, our results suggest that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator have comparable effects on repressibility. The symmetry of this interaction justified reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus (5')CTGTATAT. Although the first four positions of this sequence were most important, the last four were well conserved among binding sites and appeared to modulate repressor affinity. The role of the terminal-nucleotide contacts and the mechanism by which the internal sequences affected repressor binding are discussed.

Bacterial Proteins↗
PubMed1985

A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor.

We describe a new protein that binds to DNA and activates gene transcription in yeast. This protein, LexA-GAL4, is a hybrid of LexA, an Escherichia coli repressor protein, and GAL4, a Saccharomyces cerevisiae transcriptional activator. The hybrid protein, synthesized in yeast, activates transcription of a gene if and only if a lexA operator is present near the transcription start site. Thus, the DNA binding function of GAL4 can be replaced with that of a prokaryotic repressor without loss of the transcriptional activation function. These results suggest that DNA-bound LexA-GAL4 and DNA-bound GAL4 activate transcription by contacting other proteins.

Bacterial Proteins↗
PubMed1990

Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model.

The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond. In vitro. LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of [3H]DFP incorporation. Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated [3H]DFP at a rate indistinguishable from the intact protein. These data further support our current model for the mechanism of autodigestion and the organization of LexA.

Amino Acid Sequence↗
PubMed1990

LexA-independent expression of a mutant mucAB operon.

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.

Bacterial Proteins↗
PubMed1982

The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters.

We have found that the uvrB gene of Escherichia coli is transcribed from at least two promoters, which we call P1 and P2. Transcription from P1 begins with an ATP at +1, and transcription from P2 begins primarily with a GTP at -31. A binding site for the lexA protein (LEXA), located between the -35 sequence and Pribnow box of P2, regulates transcription from this promoter. In vitro, LEXA inhibits transcription from P2 but has no detectable effect on transcription from P1. A third promoter, P3, was also detected at -341; transcription from P3 is toward uvrB but terminates in vitro in the region of the LEXA binding site. The binding of LEXA to P2 inhibits transcription from the P3 promoter even though several hundred nucleotides separate the two promoters. The data suggest that a transcribing RNA polymerase stalls when it reaches the repressor-operator complex but remains bound to the DNA, causing a jamming of RNA polymerases between P3 and the repressor-operator complex at P2. The physiological significance of P3 is unknown.

Bacterial Proteins↗
PubMed2001

Crystal structure of LexA: a conformational switch for regulation of self-cleavage.

LexA repressor undergoes a self-cleavage reaction. In vivo, this reaction requires an activated form of RecA, but it occurs spontaneously in vitro at high pH. Accordingly, LexA must both allow self-cleavage and yet prevent this reaction in the absence of a stimulus. We have solved the crystal structures of several mutant forms of LexA. Strikingly, two distinct conformations are observed, one compatible with cleavage, and the other in which the cleavage site is approximately 20 A from the catalytic center. Our analysis provides insight into the structural and energetic features that modulate the interconversion between these two forms and hence the rate of the self-cleavage reaction. We suggest RecA activates the self-cleavage of LexA and related proteins through selective stabilization of the cleavable conformation.

Amino Acid Sequence↗
PubMed1993

DNA sequence determinants of LexA-induced DNA bending.

The LexA repressor from Escherichia coli induces DNA bending upon interaction with the two overlapping operators which regulate the transcription of the colicin A encoding gene caa. Both caa operators harbor T-tracts adjacent to their recognition motifs. These tracts have been suggested to be especially favorable for the promotion of LexA-induced DNA bending. Here we show that this is indeed the case, since disruption of the TTTT-tract adjacent to operator O1 by the replacement of the two central thymine bases by AA, GA or CG markedly reduces LexA-induced DNA bending. Simple A.T-richness in this position is thus not sufficient to promote full LexA-induced bending, albeit a TAAT sequence is always more efficient to promote bending than those sequences containing one or two C/G base pairs.

Bacterial Proteins↗
PubMed1989

Investigation of RecA--polynucleotide interactions from the measurement of LexA repressor cleavage kinetics. Presence of different types of complex.

The proteolysis of the LexA repressor in the presence of RecA and various polynucleotides was studied by measuring the fluorescence decrease of LexA upon cleavage. The results were compared with the DNA binding of RecA to investigate the presence of multiple DNA-RecA complexes. All single-stranded polydeoxyribonucleotides (DNA) efficiently stimulated the proteolysis and the maximum activation was reached in the presence of three or four nucleotides of polynucleotide per monomer of RecA. The stimulative effect was decreased in the presence of larger amounts of poly(dA), poly(dT) or heat-denatured DNA, whereas the excess of single-stranded DNAs chemically modified with chloroacetaldehyde did not present such an inhibitory effect, despite the fact that a second DNA molecule is likely to interact with RecA as monitored by the intrinsic fluorescence of these DNA species. The complicated cleavage promotion and inhibition pattern is tentatively explained by a three-state model assuming that RecA may interact with three single-stranded DNA molecules. According to this model, occupation of the first site would be necessary and sufficient for cleavage promotion, the second site would be neutral with respect to cleavage and the occupation of the third site would inhibit LexA cleavage at least partially. Double-stranded natural DNA did not stimulate cleavage, even under conditions where RecA binds quantitatively to the DNA. No polyribonucleotides (RNA) examined showed a significant stimulative effect either, nor did they appear to interact with RecA.

Bacterial Proteins↗
PubMed2002

DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexA.

The ubiquitous and highly conserved RecA protein is generally expressed from a single promoter, which is regulated by LexA in conjunction with RecA. We show here using transcriptional fusions to a reporter gene that the Mycobacterium tuberculosis recA gene is expressed from two promoters. Although one promoter is clearly regulated in the classical way, the other remains DNA damage inducible in the absence of RecA or when LexA binding is prevented. These observations demonstrate convincingly for the first time that there is a novel mechanism of DNA damage induction in M. tuberculosis that is independent of LexA and RecA.

Amino Acid Sequence↗
PubMed2000

H2O2-induced cross-protection against UV-C killing in Escherichia coli is blocked in a lexA (Def) background.

Pretreatment with 2.5 mM H2O2 protects E. coli cells against UV-C killing, a phenomenon independent of LexA cleavage. In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain [lexA (Ind-)]. Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response.

Bacterial Proteins↗
PubMed1988

Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli.

We compared the biochemical properties of the RecA441 protein to those of the wild-type RecA protein in an effort to account for the constitutive protease activity observed in recA441 strains. The two RecA proteins have similar properties in the absence of single-stranded DNA binding protein (SSB protein), and the differences that do exist shed little light on the temperature-inducible phenotype observed in recA441 strains. In contrast, several biochemical differences are apparent when the two proteins are compared in the presence of SSB protein, and these are conducive to a hypothesis that explains the temperature-sensitive behavior observed in these strains. We find that both the single-stranded DNA (ssDNA)-dependent ATPase and LexA-protease activities of RecA441 protein are more resistant to inhibition by SSB protein than are the activities of the wild-type protein. Additionally, the RecA441 protein is more capable of using ssDNA that has been precoated with SSB protein as a substrate for ATPase and protease activities, implying that RecA441 protein is more proficient at displacing SSB protein from ssDNA. The enhanced SSB protein displacement ability of the RecA441 protein is dependent on elevated temperature. These observations are consistent with the hypothesis that the RecA441 protein competes more efficiently with SSB protein for limited ssDNA sites and can be activated to cleave repressors at elevated temperature by displacing SSB protein from the limited ssDNA that occurs naturally in Escherichia coli. Neither the ssDNA binding characteristics of the RecA441 protein nor the rate at which it transfers from one DNA molecule to another provides an explanation for its enhanced activities, leading us to conclude that kinetics of RecA441 protein association with DNA may be responsible for the properties of the RecA441 protein.

Adenosine↗
PubMed2006

Characterization of the global transcriptional responses to different types of DNA damage and disruption of replication in Bacillus subtilis.

DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p-hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICEBs1) and resident prophages (PBSX and SPbeta), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA. Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.

Bacillus Phages↗
PubMed2003

A cka-gfp transcriptional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population.

In prokaryotes, only a few examples of differential gene expression in cell populations have been described. Colicin production in natural populations of Escherichia coli, while providing a competitive advantage in the natural habitat, also leads to lysis of the toxin-producing cell. Colicin K synthesis has been found to be induced due to an increase in ppGpp (I. Kuhar, J. P. van Putten, D. Zgur-Bertok, W. Gaastra, and B. J. Jordi, Mol. Microbiol. 41:207-216). Using two transcriptional fusions, cka-gfp and cki-gfp, we show that at the single-cell level, the colicin K activity gene cka is expressed in only 3% of the bacterial population upon induction by nutrient starvation. In contrast, the immunity gene cki is expressed in the large majority of the cells. Expression of the cka-gfp fusion in a lexA-defective strain and in a relA spoT mutant strain indicates that differential expression of cka is established primarily at the level of transcription.

Bacterial Proteins↗
PubMed2000

Identification of additional genes belonging to the LexA regulon in Escherichia coli.

Exposure of Escherichia coli to a variety of DNA-damaging agents results in the induction of the global 'SOS response'. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA-regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5'-TACTG(TA)5CAGTA-3'. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA-regulated genes. These searches identified a total of 69 potential LexA-regulated genes/operons with a heterology index of <15 and included all previously characterized LexA-regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage-inducible gene expression in a wild-type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non-inducible lexA(Ind-) cell. These experiments have allowed us to identify seven new LexA-regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA-regulated gene is discussed.

Amino Acid Sequence↗