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At least 199 records · Page 11Linked to original sources
PubMed1988

Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination.

The ruv gene of Escherichia coli, which is involved in DNA repair and recombination, was cloned on a plasmid vector. The DNA of the ruv region was sequenced; it had two open reading frames in tandem that could code for 22- and 37-kilodalton proteins. The proteins encoded by these open reading frames were identified by the maxicell method. The two genes were aligned in the same orientation and regulated by the SOS system, so the two genes probably constitute an operon. The distal one complemented the ruv mutations. Transcription of the operon was studied both in vivo and in vitro. Two transcription initiation sites were identified upstream of the coding frames, and the transcription from both sites was repressed by the LexA repressor. A DNA sequence that is homologous to the SOS box and bound by LexA protein was found in the regulatory region of the operon. The amino acid sequence of Ruv protein deduced from the DNA sequence shows a high degree of homology to the consensus sequence shared by ATP-binding proteins.

Amino Acid Sequence↗
PubMed1994

Targeting of promoters for trans activation by a carboxy-terminal domain of the NS-1 protein of the parvovirus minute virus of mice.

The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.

Animals↗
PubMed1993

Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites.

We used the interaction trap to isolate a novel human protein that specifically interacts with Max. This protein, Mxi1 (for Max interactor 1), contains a bHLH-Zip motif that is similar to that found in Myc family proteins. Mxi1 interacts specifically with Max to form heterodimers that efficiently bind to the Myc-Max consensus recognition site. When bound to DNA by a LexA moiety in yeast, Mxi1 does not stimulate transcription. mxi1 mRNA is expressed in many tissues, and its expression is elevated in U-937 myeloid leukemia cells that have been stimulated to differentiate. These facts are consistent with a model in which Mxi1-Max heterodimers indirectly inhibit Myc function in two ways: first, by sequestering Max, thus preventing the formation of Myc-Max heterodimers, and second, by competing with Myc-Max heterodimers for binding to target sites.

Amino Acid Sequence↗
PubMed1991

Novel SOS phenotypes caused by second-site mutations in the recA430 gene of Escherichia coli.

E coli recA430 mutants are recombination-proficient, extremely UV sensitive, UV nonmutable and partially deficient in RecA-mediated proteolysis and in RecA-dependent 'induced replisome reactivation' (IRR), the ability to recover DNA replication activity after UV irradiation. To determine how this pleiotropic phenotype can be altered by mutation, we isolated 10 independent derivatives of a recA430 strain, selecting for increased UV resistance. Eight of the 10 owed their resistance to altered recA alleles. We here describe the phenotypes conferred by two of the new recA alleles (recA720 and recA727), each of which contains the original recA430 mutation (G662 to A) and a second-site transition: T167 to C in recA720, and G103 to A in recA727. The second-site change in recA720 suppresses all the defects caused by recA430, and causes RecA720 to exhibit greater activity than RecA+ in some respects. Some, but not all, of the recA430 defects are partially corrected by the second-site mutation in recA727.

Bacterial Proteins↗
PubMed2006

The stability of transmembrane helix interactions measured in a biological membrane.

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.

Amino Acid Sequence↗
PubMed1996

Reexamining interaction of the SH2 domains of SYP and GAP with insulin and IGF-1 receptors in the two-hybrid system.

Direct interaction of effector proteins such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), SYP (SH2-domain-containing tyrosine phosphatase) and GAP (Ras-GTPase activating protein) with the insulin receptor (IR) and insulin-like growth factor-1 (IGF-1) type 1 receptor (IGF-1R) has been reported in some studies. Interaction of SYP and GAP with IR and IGF-1R was re-investigated here in the two-hybrid system by assessing his3/lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic beta domain of IR and IGF-1R and various SH2-subdomains of SYP and GAP. None of the subdomains of SYP and GAP tested were able to activate his3/lacZ, whereas these reporter genes were strongly activated when p85 was used as we have recently shown. Thus, interaction of SYP and GAP with IR and IGF-1R, if any, would be weak and/or transient as compared to that of p85.

Bacterial Proteins↗
PubMed2002

Evaluation of thresholds for the detection of binding sites for regulatory proteins in Escherichia coli K12 DNA.

BACKGROUND: Sites in DNA that bind regulatory proteins can be detected computationally in various ways. Pattern discovery methods analyze collections of genes suspected to be co-regulated on the evidence, for example, of clustering of transcriptome data. Pattern searching methods use sequences with known binding sites to find other genes regulated by a given protein. Such computational methods are important strategies in the discovery and elaboration of regulatory networks and can provide the experimental biologist with a precise prediction of a binding site or identify a gene as a member of a set of co-regulated genes (a regulon). As more variations on such methods are published, however, thorough evaluation is necessary, as performance may differ depending on the conditions of use. Detailed evaluation also helps to improve and understand the behavior of the different methods and computational strategies. RESULTS: We used a collection of 86 regulons from Escherichia coli as datasets to evaluate two methods for pattern discovery and pattern searching: dyad analysis/dyad sweeping using the program Dyad-analysis, and multiple alignment using the programs Consensus/Patser. Clearly defined statistical parameters are used to evaluate the two methods in different situations. We placed particular emphasis on minimizing the rate of false positives. CONCLUSIONS: As a general rule, sensors obtained from experimentally reported binding sites in DNA frequently locate true sites as the highest-scoring sequences within a given upstream region, especially using Consensus/Patser. Pattern discovery is still an unsolved problem, although in the cases where Dyad-analysis finds significant dyads (around 50%), these frequently correspond to true binding sites. With more robust methods, regulatory predictions could help identify the function of unknown genes.

Bacterial Proteins↗
PubMed2003

Simultaneous cloning of open reading frames into several different expression vectors.

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.

Bacterial Proteins↗
PubMed1985

Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction.

The products of the lexA and recA genes play central roles in the regulation of the Escherichia coli SOS response. We have measured the rate of mRNA synthesis from each gene at intervals following various inducing treatments in order to obtain a more precise timing of the induction process. Further, we provide quantitative evidence for kinetics of decay from fully induced levels of mRNA synthesis to basal levels as the cells shut down the SOS response which are in agreement with previously published data on the expression of specific SOS functions. The induction kinetics of lexA and recA gene expression are parallel except for nalidixic acid (NAL) treatment, with the actual levels of lexA mRNA synthesis being about 10-fold lower than that of recA. Reestablishment of repression from RecA commenced over 30 min earlier than from lexA. These results are fully consistent with the model that the functions result from the increased gene expression.

Bacterial Proteins↗
PubMed1999

Dynamics of the inducing signal for the SOS regulatory system in Escherichia coli after ultraviolet irradiation.

SOS response in Escherichia coli is induced by various DNA-damaging treatments, for example by ultraviolet irradiation, to help a cell to recover from the damage. During induction of the SOS regulatory system, generation of the inducing signal for the system is the early step. In the present study a model for quantitative description of the signal dynamics is developed. We derive the inducing signal, in terms of concentration of single-stranded DNA, as a function of time since the moment of ultraviolet irradiation. Simulation of the signal level after irradiation with two doses of 5 and 20 J m-2 is presented. This provides quantitative description of the event that controls various cellular physiological reactions induced in the course of the SOS response. The dynamics of the signal level are then used as an input for a dynamical equation description of the SOS regulatory system that we proposed earlier. This allows for a quantitative analysis of the subsequent step in the SOS induction: cleavage of LexA protein, a negative regulator of the SOS system. The model is verified against available experimental data for LexA protein level in ultraviolet radiation-induced Escherichia coli cells.

Bacterial Proteins↗
PubMed1987

Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.

Amino Acid Sequence↗
PubMed1992

Activation of recA protein. The open helix model for LexA cleavage.

RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.

Adenosine Triphosphate↗
PubMed2006

[SOS-induction in the presence of the plasmid pKM101 in the bacterial cells Escherichia coli K12].

Induction of transcription by the plasmid pKM101 (mutability mediating derivate of the plasmid R46) of the sfiA gene controlling cell division and of the fruA gene encoding the fructose specific enzyme II of the phosphoenolpyruvate-phosphotransferase system in intact cultures of Escherichia coli was studied. The genes under study were fused to the bacteriophage Mu dl (Ap lac). Activation of the sfiA gene, a typical member of the SOS-regulon, was demonstrated to depend on the key genes of the SOS-system-recA and lexA. In contrast, the fruA gene that is non-inducible by the UV-light, a classical SOS-inducing agent, is not activated by the presence of the plasmid pKMIO1 in the bacterial cells. The data obtained suggest that the presence of pKMIO1 plasmid in the Escherichia coli cells induces a SOS-signal as a consequence of the plasmid DNA replication or its conjugative transfer.

Bacterial Proteins↗
PubMed1988

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli.

Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ+. dnaQ49 is a recessive allele that confers temperature-sensitive and salt-suppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.

Alleles↗
PubMed1997

A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators.

The SIN3 gene is required for the transcriptional repression of diverse genes in Saccharomyces cerevisiae. Sin3p does not bind directly to DNA but is thought to be targeted to promoters by interacting with sequence-specific DNA-binding proteins. We show here that Sin3p is present in a large multiprotein complex with an apparent molecular mass, estimated by gel filtration chromatography, of greater than 2 million Da. Genetic studies have shown that the yeast RPD3 gene has a function similar to that of SIN3 in transcriptional regulation, as SIN3 and RPD3 negatively regulate the same set of genes. The SIN3 and RPD3 genes are conserved from yeasts to mammals, and recent work suggests that RPD3 may encode a histone deacetylase. We show that Rpd3p is present in the Sin3p complex and that an rpd3 mutation eliminates SIN3-dependent repression. Thus, Sin3p may function as a bridge to recruit the Rpd3p histone deacetylase to specific promoters.

Bacterial Proteins↗
PubMed1991

Identification of dinR, a DNA damage-inducible regulator gene of Bacillus subtilis.

A Bacillus subtilis strain deficient in homologous recombination was isolated from a library of Tn917lac insertion mutants. The interrupted locus consists of an open reading frame encoding a 22,823-dalton polypeptide. Analysis of the deduced amino acid sequence revealed 34% identity and 47.3% similarity with the LexA protein from Escherichia coli. The gene was designated dinR. It is located between the recA and thyA genetic markers, at 162 degrees on the B. subtilis chromosome. The dinR gene was shown to be expressed during the entire B. subtilis cellular cycle with at least a threefold increase when cells develop competence. In addition, the use of a merodiploid strain, in which a copy of the wild-type dinR gene coexists with a dinR-lacZ transcriptional fusion, demonstrated that dinR is an SOS gene and that the SOS-induced expression of dinR occurred only when a wild-type copy of dinR was present. In addition, DinR seems to regulate the expression of dinC, another SOS gene.

Amino Acid Sequence↗