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PubMed2006

Modulation of RNA polymerase core functions by basal transcription factor TFB/TFIIB.

The archaeal basal transcriptional machinery consists of TBP (TATA-binding protein), TFB (transcription factor B; a homologue of eukaryotic TFIIB) and an RNA polymerase that is structurally very similar to eukaryotic RNA polymerase II. This constellation of factors is sufficient to assemble specifically on a TATA box-containing promoter and to initiate transcription at a specific start site. We have used this system to study the functional interaction between basal transcription factors and RNA polymerase, with special emphasis on the post-recruitment function of TFB. A bioinformatics analysis of the B-finger of archaeal TFB and eukaryotic TFIIB reveals that this structure undergoes rapid and apparently systematic evolution in archaeal and eukaryotic evolutionary domains. We provide a detailed analysis of these changes and discuss their possible functional implications.

Amino Acid Sequence↗

PubMed2006

Evolution of the soluble nitrate reductase: defining the monomeric periplasmic nitrate reductase subgroup.

Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.

Bacterial Proteins↗

PubMed2001

Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages.

Phage-host interactions remain poorly understood in lactic acid bacteria and essentially in all Gram-positive bacteria. The aim of this study was to identify the phage genetic determinant (anti-receptor) involved in the recognition of Streptococcus thermophilus hosts. The complete genomic sequence of the lytic S. thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti-receptor gene. The orf18 of six additional S. thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1. The deduced ORF18 was divided into three domains. The first domain, which contains the N-terminal part of the protein, was conserved in all seven phages. The second domain was detected in only two phages and flanked by a motif called collagen-like repeats. The second domain also contained a variable region (VR1). All seven phages had a third domain that consisted of the C-terminal section of the protein as well as another variable region (VR2). Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4. All DT1 chimeric phages acquired the host range of phage MD4. Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2. This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram-positive bacteria.

Adsorption↗

PubMed2003

Microarray expression profiling: capturing a genome-wide portrait of the transcriptome.

The bacterial transcriptome is a dynamic entity that reflects the organism's immediate, ongoing and genome-wide response to its environment. Microarray expression profiling provides a comprehensive portrait of the transcriptional world enabling us to view the organism as a 'system' that is more than the sum of its parts. The vigilance of microorganisms to environmental change, the alacrity of the transcriptional response, the short half-life of bacterial mRNA and the genome-scale nature of the investigation collectively explain the power of this method. These same features pose the most significant experimental design and execution issues which, unless surmounted, predictably generate a distorted image of the transcriptome. Conversely, the expression profile of a properly conceived and conducted microarray experiment can be used for hypothesis testing: disclosure of the metabolic and biosynthetic pathways that underlie adaptation of the organism to chang-ing conditions of growth; the identification of co-ordinately regulated genes; the regulatory circuits and signal transduction systems that mediate the adaptive response; and temporal features of developmental programmes. The study of bacterial pathogenesis by microarray expression profiling poses special challenges and opportunities. Although the technical hurdles are many, obtaining expression profiles of an organism growing in tissue will probably reveal strategies for growth and survival in the host's microenvironment. Identifying these colonization strategies and their cognate expression patterns involves a 'deconstruction' process that combines bioinformatics analysis and in vitro DNA array experimentation.

Bacteria↗

PubMed2002

Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis.

In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al. Scand J Immunol 1999;50:510-8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione-S-transferase (GST) fusion proteins in recombinant Escherichia coli. Polyclonal antibodies against a predicted B-cell epitope of each of the Mce1 proteins of M. tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin. These antibodies reacted specifically with the corresponding fusion protein, except for GST-Mce1F. A mouse monoclonal antibody, TB1-5 76C, raised against a synthetic 60-mer peptide corresponding to the residues 106-165 in the N-terminal part of Mce1A, reacted strongly with GST-Mce1A. The antibody cross-reacted with GST-Mce1F, but not with the other recombinant GST-Mce1 fusion proteins or free GST. Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains. Higher homology was found between the residues 106-165 of Mce1A and the residues 347-406, further into the mature Mce1F polypeptide chain. There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1-5 76C may be narrowed to the KRRITPKD region, the residues 131-138 in Mce1A corresponding to the residues 372-379 in Mce1F. This was confirmed in enzyme-linked immunosorbent assay, showing binding of TB1-5 76C to a 17-mer synthetic peptide containing the KRRITPKD sequence.

Amino Acid Sequence↗

PubMed2003

Molecular characterization of Enterococcus faecalis two-component signal transduction pathways related to environmental stresses.

A key issue in the comprehension of the Enterococcus faecalis stress response is to understand how this bacterium is able to modulate its gene expression in accordance to environmental conditions. Through bioinformatic analysis of the E. faecalis V583 genome, nine two-component systems and a single orphan response regulator were identified. A transcriptional study gave evidence of four systems whose expression is induced by at least one environmental stress. In addition, gene disruption experiments allowed the isolation of eight response regulator mutants. Insertional inactivation of the response regulator gene err 05 resulted in growth default and cell morphology alterations; and also in expression default of the sagA gene, this latter being recently shown involved in E. faecalis stress resistance toward numerous lethal treatments (Y. Le Breton, A. Mazé, A. Hartke, S. Lemarinier, Y. Auffray and A. Rincé (2002) Current Microbiol 45: 434-439). Disruption of the err 04, err 08 or err 18 genes resulted in sensitivity towards heat. Finally, the err 10 mutant was shown to be more sensitive to acid pH and NaCl whereas its growth was less affected by bile salts or heat. We also demonstrated that the heat resistance phenotype of the err 10 mutant was correlated with an increase of the heat shock proteins DnaK and GroEL level.

Amino Acid Sequence↗

PubMed2005

Rapid hyperosmotic coinduction of two tilapia (Oreochromis mossambicus) transcription factors in gill cells.

Gills of euryhaline teleosts are excellent models for studying osmotic-stress adaptation because they directly contact the aquatic environment and are an important effector tissue during osmotic stress. We acclimated tilapia (Oreochromis mossambicus) from fresh water (FW) to seawater (SW); performed suppression subtractive hybridization of gill mRNAs; and identified two transcription factors, osmotic stress transcription factor 1 (OSTF1) and the tilapia homolog of transcription factor II B (TFIIB), that are rapidly and transiently induced during hyperosmotic stress. mRNA levels increase 6-fold for OSTF1 and 4-fold for TFIIB, and they reach maxima 2 h after SW transfer. Protein levels increase 7.5-fold for OSTF1 and 9-fold for TFIIB, and they reach maxima 4 h after SW transfer. Induction of OSTF1 and TFIIB increases gradually with increasing salinity. Induction of OSTF1 and TFIIB is specific for osmotic stress and absent during oxidative stress (1 mM H2O2) or heat shock (+10 degrees C). Bioinformatic analysis of OSTF1 reveals that it is a transcription factor of the TGF-beta-stimulated clone 22/GILZ family. Because some mammalian homologs are strongly induced by glucocorticoids, OSTF1 may represent the molecular link between the SW hormone cortisol and transcriptional regulation of ion transport and cell differentiation in teleost gills. Coinduction of OSTF1 and TFIIB may serve to recruit TFIIB preferentially to OSTF1 target genes during hyperosmotic stress and compensate for reduced rates of transcription resulting from salt-induced chromatin compaction. We conclude that OSTF1 and TFIIB are critical elements of osmosensory signal transduction in euryhaline teleosts that mediate osmotic adaptation by means of transcriptional regulation.

Adaptation, Physiological↗

PubMed2005

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2.

During infection of Arabidopsis thaliana, the bacterium Pseudomonas syringae pv tomato delivers the effector protein AvrRpt2 into the plant cell cytosol. Within the plant cell, AvrRpt2 undergoes N-terminal processing and causes elimination of Arabidopsis RIN4. Previous work established that AvrRpt2 is a putative cysteine protease, and AvrRpt2 processing and RIN4 elimination require an intact predicted catalytic triad in that AvrRpt2. In this work, proteolytic events that depend on AvrRpt2 activity were characterized. The amino acid sequence surrounding the processing site of AvrRpt2 and two related sequences from RIN4 triggered Avr-Rpt2-dependent proteolytic cleavage of a synthetic substrate, demonstrating that these sequences are cleavage recognition sites for AvrRpt2 activity. Processing-deficient AvrRpt2 mutants were identified and shown to retain their ability to eliminate wild-type RIN4. Single amino acid substitutions were made in each of the two RIN4 cleavage sites, and mutation of both sites resulted in cleavage-resistant RIN4. Growth of Pseudomonas expressing AvrRpt2 was significantly higher than catalytically inactive mutants on Arabidopsis rin4/rps2 mutant plants, suggesting there are additional protein targets of AvrRpt2 that account for the virulence activity of this effector. Bioinformatics analysis identified putative Arabidopsis proteins containing sequences similar to the proteolytic cleavage sites conserved in AvrRpt2 and RIN4. Several of these proteins were eliminated in an AvrRpt2-dependent manner in a transient in planta expression system. These results identify amino acids important for AvrRpt2 substrate recognition and cleavage as well as demonstrate AvrRpt2 protease activity eliminates multiple Arabidopsis proteins in a transient expression system.

Amino Acid Sequence↗

PubMed2005

Deleted in liver cancer 2 (DLC2) suppresses cell transformation by means of inhibition of RhoA activity.

The deleted in liver cancer 2 (DLC2) gene, located at chromosome 13q12.3, is a recently identified tumor suppressor gene. The gene is frequently underexpressed in human hepatocellular carcinoma, and its chromosomal region shows frequent deletion. DLC2 encodes a unique RhoGTPase-activating protein (RhoGAP) specific for small RhoGTPases, RhoA, and Cdc42. With bioinformatic analysis, we have identified four different isoforms of DLC2, which we named DLC2alpha, DLC2beta, DLC2gamma, and DLC2delta. Three of the isoforms contain the RhoGAP domain, namely, DLC2alpha, DLC2beta, and DLC2gamma. Ectopic expression of these three isoforms in mouse fibroblasts showed cytoplasmic localization. Of interest, overexpression of these isoforms suppressed the lysophosphatidic acid-induced stress fiber formation in mouse fibroblasts and changed the morphology of the transfected cells from angular and spindle to round. Furthermore, the RhoA pull-down assay demonstrated a remarkable reduction in RhoA activity in the DLC2 transiently transfected cells. In contrast, cells transfected with inactive DLC2 GAP-mutant remained unchanged in cell morphology, actin stress fiber formation, and RhoA activity. HepG2 hepatoma cells stably transfected with the DLC2gamma isoform also changed to a round morphology, as in mouse fibroblasts. Of significance, these DLC2gamma stable transfectants showed marked suppression in cell proliferation, motility, and transformation, and there was a remarkable reduction in in vivo RhoA activity in these cells. These results suggest that DLC2 exhibits its tumor suppressor functions in vivo as a GAP specific for RhoA, exerting its effects in suppression of cytoskeleton reorganization, cell growth, cell migration, and transformation.

3T3 Cells↗

PubMed2005

MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.

Animals↗

PubMed2006

Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides.

Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-beta-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7.

Adenosine Triphosphate↗

PubMed2006

PlyC: a multimeric bacteriophage lysin.

Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Gram-positive lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique lysin from the streptococcal bacteriophage C(1), termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases.

Cell Wall↗

PubMed2006

A FoxO-Smad synexpression group in human keratinocytes.

Transforming growth factor beta (TGF-beta) signals through activation of Smad transcription factors. Activated Smad proteins associate with different DNA-binding cofactors for the recognition and regulation of specific target genes. Members of the forkhead box O family (FoxO1, FoxO3, and FoxO4) play such a role in the induction of the cyclin-dependent kinase inhibitors p15Ink4b and p21Cip1. To delineate the organization of the TGF-beta response in human keratinocytes, we defined the set of genes whose activation by TGF-beta requires both FoxO and Smad functions. FoxO factors are shown to be essential for 11 of the 115 immediate gene activation responses to TGF-beta in these cells. FoxO1, FoxO3, and FoxO4 act redundantly as mediators of these effects. Smad4, which functions as a partner of receptor-phosphorylated Smad2/3, is required for all of these responses. These results define a FoxO-Smad synexpression group or group of genes that are jointly induced by a common mechanism in response to TGF-beta. In addition to p15INK4b and p21CIP1, these genes include mediators of stress responses (GADD45A, GADD45B, and IER1) and adaptive cell signaling responses (CTGF, JAG1, LEMD3, SGK, CDC42EP3, and OVOL1). Bioinformatic analysis of the promoter region of these genes reveals diverse configurations of Smad and FoxO binding elements, implying differences in the regulatory properties of this group of genes. Indeed, a subset of FoxO/Smad-dependent TGF-beta gene responses additionally require the transcription factor CCAAT/enhancer-binding protein beta. The composition of the FoxO-Smad synexpression group suggests that stress reactions and adaptive functions accompany the cytostatic response of keratinocytes to TGF-beta.

Binding Sites↗

PubMed2000

Gene expression profiling in the human hypothalamus-pituitary-adrenal axis and full-length cDNA cloning.

The primary neuroendocrine interface, hypothalamus and pituitary, together with adrenals, constitute the major axis responsible for the maintenance of homeostasis and the response to the perturbations in the environment. The gene expression profiling in the human hypothalamus-pituitary-adrenal axis was catalogued by generating a large amount of expressed sequence tags (ESTs), followed by bioinformatics analysis (http://www.chgc.sh.cn/ database). Totally, 25,973 sequences of good quality were obtained from 31,130 clones (83.4%) from cDNA libraries of the hypothalamus, pituitary, and adrenal glands. After eliminating 5,347 sequences corresponding to repetitive elements and mtDNA, 20,626 ESTs could be assembled into 9, 175 clusters (3,979, 3,074, and 4,116 clusters in hypothalamus, pituitary, and adrenal glands, respectively) when overlapping ESTs were integrated. Of these clusters, 2,777 (30.3%) corresponded to known genes, 4,165 (44.8%) to dbESTs, and 2,233 (24.3%) to novel ESTs. The gene expression profiles reflected well the functional characteristics of the three levels in the hypothalamus-pituitary-adrenal axis, because most of the 20 genes with highest expression showed statistical difference in terms of tissue distribution, including a group of tissue-specific functional markers. Meanwhile, some findings were made with regard to the physiology of the axis, and 200 full-length cDNAs of novel genes were cloned and sequenced. All of these data may contribute to the understanding of the neuroendocrine regulation of human life.

Alternative Splicing↗

PubMed2002

The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract.

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gastrointestinal tract (GIT) and vagina. Although not numerically dominant in the complex intestinal microflora, they are considered as key commensals that promote a healthy GIT. We determined the 2.26-Mb genome sequence of an infant-derived strain of Bifidobacterium longum, and identified 1,730 possible coding sequences organized in a 60%-GC circular chromosome. Bioinformatic analysis revealed several physiological traits that could partially explain the successful adaptation of this bacteria to the colon. An unexpectedly large number of the predicted proteins appeared to be specialized for catabolism of a variety of oligosaccharides, some possibly released by rare or novel glycosyl hydrolases acting on "nondigestible" plant polymers or host-derived glycoproteins and glycoconjugates. This ability to scavenge from a large variety of nutrients likely contributes to the competitiveness and persistence of bifidobacteria in the colon. Many genes for oligosaccharide metabolism were found in self-regulated modules that appear to have arisen in part from gene duplication or horizontal acquisition. Complete pathways for all amino acids, nucleotides, and some key vitamins were identified; however, routes for Asp and Cys were atypical. More importantly, genome analysis provided insights into the reciprocal interactions of bifidobacteria with their hosts. We identified polypeptides that showed homology to most major proteins needed for production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the GIT. We also found a eukaryotic-type serine protease inhibitor (serpin) possibly involved in the reported immunomodulatory activity of bifidobacteria.

Adaptation, Physiological↗

PubMed2001

The plant oncogene rolD encodes a functional ornithine cyclodeaminase.

The plant oncogene rolD stimulates the reproductive phase transition in plants. We define here the function of its gene product. We show that the RolD protein bears sequence homology with ornithine cyclodeaminase, an uncommon enzyme of specialized-niche eubacteria and archaea that catalyzes the unusual NAD(+)-dependent conversion of ornithine to proline. To confirm the prediction of the bioinformatic analysis, the RolD protein was expressed in Escherichia coli and purified. An ornithine-dependent NAD(+) reduction that can be ascribed only to ornithine cyclodeaminase (OCD) activity was detected both in bacterial extracts containing RolD and in assays on the purified RolD protein. Furthermore, OCD activity was observed in soluble extracts from plants overexpressing rolD. The role of rolD in plant pathogenesis and its effect on plant reproductive development are discussed in light of the newly demonstrated enzymatic activity of its gene product.

Amino Acid Sequence↗

PubMed2000

HDAC1, a histone deacetylase, forms a complex with Hus1 and Rad9, two G2/M checkpoint Rad proteins.

HDAC1 is a member of the histone deacetylase family, which plays an important role in modulating the eukaryotic chromatin structure. Numerous studies have demonstrated its involvement in transcription and in tumorigenesis. To better understand the functions and regulation of HDAC1, a yeast two-hybrid screening approach was chosen to identify novel interactions involving HDAC1. Human HDAC1 was found to interact specifically in yeast, mammalian cells, and in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G(2)/M checkpoint control. Both HDAC1 and Hus1 proteins localize to the nuclei. Furthermore, HDAC1 and Hus1 were found to exist in a complex with Rad9, a known Hus1-interacting factor. In addition, bioinformatics analysis of the protein sequences of Hus1, Rad1, and Rad9, three checkpoint Rad proteins that form a complex, revealed that they all contain a putative proliferating cell nuclear antigen (PCNA) fold, raising the possibility that these factors may bind to DNA in a PCNA-like ring structure. The results reported in this study strongly suggest a novel pathway involving HDAC1 in G(2)/M checkpoint control through the interaction with a functional Rad complex that may utilize a PCNA-like structure. Therefore, physically and functionally similar apparatus may function during G(2)/M checkpoint and DNA replication.

Amino Acid Sequence↗

PubMed2003

The YefM antitoxin defines a family of natively unfolded proteins: implications as a novel antibacterial target.

Although natively unfolded proteins are being observed increasingly, their physiological role is not well understood. Here, we demonstrate that the Escherichia coli YefM protein is a natively unfolded antitoxin, lacking secondary structure even at low temperature or in the presence of a stabilizing agent. This conformation of the protein is suggested to have a key role in its physiological regulatory activity. Because of the unfolded state of the protein, a linear determinant rather than a conformational one is presumably being recognized by its toxin partner, YoeB. A peptide array technology allowed the identification and validation of such a determinant. This recognition element may provide a novel antibacterial target. Indeed, a pair-constrained bioinformatic analysis facilitated the definite determination of novel YefM-YoeB toxin-antitoxin systems in a large number of bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Taken together, the YefM protein defines a new family of natively unfolded proteins. The existence of a large and conserved group of proteins with a clear physiologically relevant unfolded state serves as a paradigm to understand the structural basis of this state.

Amino Acid Sequence↗