RESUMEN
After a 37 to 10°C temperature downshift the level of translation initiation factor IF2, like that of IF1 and IF3, increases at least 3-fold with respect to the ribosomes. To clarify the mechanisms and conditions leading to cold-stress induction of infB expression, the consequences of this temperature shift on infB (IF2) transcription, infB mRNA stability and translation were analysed. The Escherichia coli gene encoding IF2 is part of the metY-nusA-infB operon that contains three known promoters (P-1, P0 and P2) in addition to two promoters P3 and P4 identified in this study, the latter committed to the synthesis of a monocistronic mRNA encoding exclusively IF2. The results obtained indicate that the increased level of IF2 following cold stress depends on three mechanisms: (i) activation of all the promoters of the operon, P-1 being the most cold-responsive, as a likely consequence of the reduction of the ppGpp level that follows cold stress; (ii) a large increase in infB mRNA half-life and (iii) the cold-shock induced translational bias that ensures efficient translation of infB mRNA by the translational apparatus of cold shocked cells. A comparison of the mechanisms responsible for the cold shock induction of the three initiation factors is also presented.
Asunto(s)
Respuesta al Choque por Frío/genética , Factor 2 Procariótico de Iniciación/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética , Frío , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , Procesamiento Proteico-Postraduccional/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Ribosomas/genéticaRESUMEN
tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.
Asunto(s)
Neoplasias de la Mama/metabolismo , Fosfoproteínas/metabolismo , ARN de Transferencia de Ácido Glutámico/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Transgénicos , Fosfoproteínas/genética , ARN de Transferencia de Ácido Glutámico/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , NucleolinaRESUMEN
Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.
Asunto(s)
Antraquinonas/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/prevención & control , Antagonistas del Ácido Fólico/farmacología , Lithospermum , Receptor ErbB-2/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Antraquinonas/aislamiento & purificación , Antraquinonas/farmacocinética , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Antagonistas del Ácido Fólico/aislamiento & purificación , Antagonistas del Ácido Fólico/farmacocinética , Humanos , Lithospermum/química , Ratones Transgénicos , Raíces de Plantas , Receptor ErbB-2/genética , Transducción de Señal , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have investigated the relationship between oligomerization in solution and DNA binding for the bacterial nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional repression. Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the notion that cooperative binding of H-NS to DNA is related to protein-protein interactions. These results emphasize the relative contributions of protein-protein interactions and substrate-dependent oligomerization in the control of gene repression operated by H-NS.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutagénesis Insercional , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Clonación Molecular , Proteínas de Unión al ADN/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Multimerización de ProteínaRESUMEN
The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN sin Sentido/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/genética , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN sin Sentido/química , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Transcripción Genética , Factores de Virulencia/genéticaRESUMEN
The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , ARN sin Sentido/metabolismo , Shigella flexneri/genética , Factores de Transcripción/genética , Transcripción Genética , Proteínas Bacterianas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Regulación hacia Abajo , Regiones Promotoras Genéticas , ARN sin Sentido/química , ARN sin Sentido/genética , Shigella flexneri/patogenicidad , Factores de Transcripción/biosíntesis , Factores de Virulencia/biosíntesis , Factores de Virulencia/genéticaRESUMEN
The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient's immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.
RESUMEN
Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. coli hns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.
RESUMEN
VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF-RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to "fish" out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5'-UUUUaGYcUuUau-3') that is the RNA-converted consensus sequence previously proposed for the VirF-DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF-RNA interaction, in particular when exposed in a stem-loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA-RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem-loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA-RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.
RESUMEN
Wheat amylase/trypsin bi-functional inhibitors (ATIs) are protein stimulators of innate immune response, with a recently established role in promoting both gastrointestinal and extra-gastrointestinal inflammatory syndromes. These proteins have been reported to trigger downstream intestinal inflammation upon activation of TLR4, a member of the Toll-like family of proteins that activates signalling pathways and induces the expression of immune and pro-inflammatory genes. In this study, we demonstrated the ability of ATI to directly interact with TLR4 with nanomolar affinity, and we kinetically and structurally characterized the interaction between these macromolecules by means of a concerted approach based on surface plasmon resonance binding analyses and computational studies. On the strength of these results, we designed an oligopeptide capable of preventing the formation of the complex between ATI and the receptor.
Asunto(s)
Técnicas Biosensibles , Proteínas de Plantas/metabolismo , Receptor Toll-Like 4/metabolismo , Triticum/metabolismo , Inhibidores de Tripsina/metabolismo , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((p)ppGpp) and in vitro transcription assays, we show that the cspA promoter is sensitive to (p)ppGpp inhibition. The (p)ppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription.
RESUMEN
During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°â10 °C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.
Asunto(s)
Escherichia coli/química , ARN Bacteriano/química , ARN Ribosómico 16S/química , ARN Ribosómico 23S/química , Aclimatación , Frío , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Regulación Bacteriana de la Expresión Génica , Operón , Fosfatos/química , Polirribosomas/química , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Subunidades Ribosómicas/química , Ribosomas/química , Temperatura , Factores de Tiempo , Transcripción GenéticaRESUMEN
Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Benzofenantridinas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Antagonistas del Ácido Fólico/uso terapéutico , Isoquinolinas/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias Basocelulares/tratamiento farmacológico , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Benzofenantridinas/efectos adversos , Benzofenantridinas/farmacología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Antagonistas del Ácido Fólico/efectos adversos , Antagonistas del Ácido Fólico/farmacología , Humanos , Isoquinolinas/efectos adversos , Isoquinolinas/farmacología , Metotrexato/farmacología , Ratones , Ratones Endogámicos , Necrosis , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Neoplasias Basocelulares/enzimología , Neoplasias Basocelulares/patología , Distribución Aleatoria , Tetrahidrofolato Deshidrogenasa/química , Carga Tumoral/efectos de los fármacosRESUMEN
Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs to a transcriptional unit containing several promoters and terminators, is enhanced after cold shock, causing a transient increase of the IF3/ribosomes ratio. Here we show that after cold shock the two less used promoters (P(T) and P(I1)) remain active and/or are activated, resulting in de novo infC transcription and IF3 synthesis. These two events are partly responsible for the stoichiometric imbalance of the IF3/ribosomes ratio that contributes to establishing the cold-shock translational bias whereby cold-shock mRNAs are preferentially translated by cold-stressed cells while bulk mRNAs are discriminated against. Analysis of the IF3 functions at low temperature sheds light on the molecular mechanism by which IF3 contributes to the cold-shock translational bias. IF3 was found to cause a strong rate increase of fMet-tRNA binding to ribosomes programmed with cold-shock mRNA, an activity essential for the rapid formation of "30S initiation complexes" at low temperature. The increased IF3/ribosome ratio occurring during cold adaptation was also essential to overcome the higher stability of 70S monomers at low temperature so as to provide a sufficient pool of dissociated 30S subunits capable of "70S initiation complex" formation. Finally, at low temperature IF3 was shown to be endowed with the capacity of discriminating against translation of non-cold-shock mRNAs by a cold-shock-specific "fidelity" function operating with a mechanism different from those previously described, insofar as IF3 does not interfere with formation of 30S initiation complex containing these mRNAs, but induces the formation of nonproductive 70S initiation complexes.
Asunto(s)
Frío , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factor 3 Procariótico de Iniciación/genética , Escherichia coli/fisiología , ARN Mensajero , Ribosomas/fisiologíaRESUMEN
Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37 degrees C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold-shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.
Asunto(s)
Frío , Proteínas de Escherichia coli/genética , Factor 1 Procariótico de Iniciación/genética , Biosíntesis de Proteínas , Transcripción Genética , Adaptación Fisiológica , Northern Blotting , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Cinética , Factor 1 Procariótico de Iniciación/metabolismo , Factor 1 Procariótico de Iniciación/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasa III/metabolismo , Proteínas Ribosómicas/metabolismo , TemperaturaRESUMEN
One of the most efficient systems of acid resistance in Escherichia coli, the gad system, is based on the coordinated action of two isoforms of glutamate decarboxylase (GadA and GadB) and of a specific glutamate/gamma-aminobutyrate antiporter (GadC). The gadA/BC genes, activated in response to acid stress and in stationary phase cells, are subjected to complex circuits of regulation involving sigma70, sigmaS, cAMP receptor protein, H-NS, EvgAS, TorRS, GadE, GadX, GadW, and YdeO. Herein, we provide evidence that the nucleoid-associated protein H-NS directly functions as repressor of gadA, one of the structural genes, and gadX, a regulatory gene encoding one of the primary activators of the gad system. Band shift and DNase I footprints reveal that H-NS indeed binds to specific sites in the promoter regions of gadA and gadX and represses the transcription of these genes both in an in vitro system and in vivo. Moreover, we show that a maltose-binding protein MalE-GadX fusion is able to stimulate the promoter activity of gadA/BC, thus indicating that GadX is by itself able to up-regulate the gad genes and that a functional competition between H-NS and GadX takes place at the gadA promoter. Altogether, our results indicate that H-NS directly inhibits gadA and gadX transcription and, by controlling the intracellular level of the activator GadX, indirectly affects the expression of the whole gad system.
Asunto(s)
Factor de Transcripción de AraC/fisiología , Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/enzimología , Glutamato Descarboxilasa/metabolismo , Factor de Transcripción de AraC/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Unión Competitiva , ADN/química , Cartilla de ADN/química , ADN Superhelicoidal , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Operón Lac , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Temperatura , Regulación hacia Arriba , beta-Galactosidasa/metabolismoRESUMEN
In the human enteropathogen Shigella transcription of virF, the primary regulator of the invasion functions, is strictly temperature-dependent and is antagonistically mediated by H-NS and FIS, which bind to specific sites on the virF promoter. Here we report on the relevance of DNA geometry to the thermoregulation of virF and demonstrate that the virF promoter hosts a major DNA bend halfway between two H-NS sites. The bent region has been mutagenized in vitro to mimic temperature-induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H-NS sites are phased-out by a half-helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H-NS with the virF promoter, as well as its in vivo and in vitro temperature-dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, the virF bending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Regiones Promotoras Genéticas/genética , Shigella/genética , Shigella/patogenicidad , Secuencia de Bases , Cartilla de ADN , Escherichia coli/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Expression of Escherichia coli hupA and hupB, the structural genes encoding the most abundant nucleoid-associated proteins HUalpha and HUbeta has been studied during cold shock. This article demonstrates that: (i) transcriptional expression of hupA is blocked following a sudden temperature downshift (from 37 degrees C to 10 degrees C), whereas transcription of hupB from the P2 and P3 promoters is maintained at a constitutive level and is activated de novo from the P4 promoter; (ii) all three hupB mRNAs (transcribed from the three natural promoters P2, P3 and P4) become much more stable than the single hupA transcript; and (iii) the hupB transcripts, unlike that of hupA, are efficiently translated in vivo during cold acclimation and can be actively translated in vitro at low temperature. Taken together, the results indicate that during cold shock the expression of the HUbeta subunit is preferentially stimulated and that of HUalpha repressed, suggesting that an altered HUalpha to HUbeta expression ratio resulting in an increase of HUalpha/HUbeta heterodimers and/or (HUbeta)2 homodimers may play an important role during cold adaptation.