RESUMEN
The ccpA gene was inactivated in the polyhydroxybutyrate (PHB)-producing strain Bacillus sp. MA3.3 in order to reduce glucose catabolite repression over pentoses and develop improved bacterial strains for the production of PHB from lignocellulosic hydrolysates. Mutant Bacillus sp. MSL7 ΔCcpA are unable to grow on glucose and ammonia as sole carbon and nitrogen sources, respectively. Supplementation of glutamate as the nitrogen source or the substitution of the carbon source by xylose allowed the mutant to partially recover its growth performance. RT-PCR showed that CcpA stimulates the expression of the operon (gltAB),responsible for ammonia assimilation via glutamate in Bacillus sp. MA3.3. Moreover, it was demonstrated that the supplementation of xylose or glutamate was capable of stimulating gltAB operon expression independently of CcpA. In PHB production experiments in mineral media, it has been observed that the glucose catabolite repression over the pentoses was partially released in MSL7. Although the carbohydrate consumption is faster in the ccpA mutant, the biomass and PHB biosynthesis are lower, even with supplementation of glutamate. This is attributed to an increase of acetyl-CoA flux towards the tricarboxylic acid cycle observed in the mutant.
Asunto(s)
Bacillus/metabolismo , Proteínas Bacterianas/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Amoníaco/metabolismo , Bacillus/genética , Bacillus/crecimiento & desarrollo , Bacillus/aislamiento & purificación , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Xilosa/metabolismoRESUMEN
The use of restricted replication-competent adenoviruses (RRCAs) inducing tumor cell-specific lysis is a promising approach in cancer gene therapy. However, the use of RRCAs in humans carries considerable risk, since after injection into the patient, further regulation or inhibition of virus replication from the outside is impossible. Therefore, we have developed a novel system allowing external pharmacological control of RRCA replication. We show here that a tumor-selective E1B-deleted RRCA can be tightly regulated by use of doxycycline (dox)-controlled adenoviral E1A gene expression, which in turn determines vector replication. RRCA replication is switched on by addition and switched off by withdrawal of dox. The system results in efficient tumor cell killing after induction by dox, whereas cells are unaffected by the uninduced system. It was also employed for efficient external control of transgene expression from cotransfected replication-deficient adenovectors. Furthermore, the use of a liver cell-specific human alpha1-antitrypsin (hAAT)-promoter driving a tetracycline-controlled transcriptional silencer allowed specific protection of cells with hAAT-promoter activity in the absence of dox in vitro and in vivo, delineating a new principle of 'tissue protective' gene therapy. The concept of external control of RRCAs may help to improve the safety of cancer gene therapy.
Asunto(s)
Adenoviridae/genética , Doxiciclina/farmacología , Terapia Genética/métodos , Neoplasias/terapia , Inhibidores de la Síntesis de la Proteína/farmacología , Replicación Viral/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/genética , Animales , Eliminación de Gen , Humanos , Ratones , Ratones Desnudos , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacosRESUMEN
Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2(S)-M2 and the other has a rtTA responsive promoter driving the expression of beta-galactosidase gene (lacZ). The rtTA2(S)-M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.
Asunto(s)
Antibacterianos/uso terapéutico , Doxiciclina/uso terapéutico , Regulación de la Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , VIH-1/genética , Animales , Encéfalo/metabolismo , Células CHO , Cricetinae , Endotelio Vascular , Vectores Genéticos/genética , Humanos , Hibridomas , Operón Lac , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores , Transducción Genética/métodosRESUMEN
Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2(s)-M2) alone or in combination with Tet-repressor (tTS-Kid) were explored in the context of helper-dependent adenovirus vector. Various genetic elements were assembled in a series of vectors and the ability to control secreted alkaline phosphatase expression evaluated in vitro in HeLa cells and in vivo by intramuscular injection in both C57/B6 and Balb/C nude mice. The results allow us to draw some general conclusions about the combination of transcription regulators and their relative orientation to the transgene to achieve maximal induction, while minimizing leakiness of expression.
Asunto(s)
Regulación de la Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/terapia , Adenoviridae/genética , Animales , Femenino , Células HeLa , Virus Helper , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , TetraciclinaRESUMEN
In addition to the originally described Tet transactivator tTA, several variants including transrepressors (tTRs) and reverse transactivators (rtTAs) have been constructed, which we employ here to establish a set of HeLa cell lines carrying different combinations of chromosomally integrated Tet transregulators. We first compare the regulatory properties of these lines using transient transfection of a luciferase reporter gene. Cell lines carrying rtTA-S2 or rtTA-M2 show reduced activity in the absence of dox and higher activation levels in its presence compared to an rtTA line. rtTA-M2 and its synthetic counterpart rtTA2S-M2 show the same regulation pattern. The replacement of the VP16 activation domain in rtTA-S2 or tTA by p65 leads to slightly reduced expression levels. Combination of an rtTA variant with the transrepressor tTR shows active repression of basal expression without affecting the activation level of the transiently transfected reporter gene. However, if the target gene is also chromosomally integrated, then tTR leads to a further reduction of basal expression and also of the maximal expression level. The results demonstrate that different regulatory windows can be achieved using various transregulators or combinations thereof. Thus, the most appropriate combination of regulators can be chosen depending on the application and cell line desired. We suspect that these properties would also allow the construction of transgenic organisms with preselected expression windows.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Tetraciclina/farmacología , Proteínas Represoras/genética , Transactivadores/genéticaRESUMEN
Since their inception, tetracycline (Tet)-inducible systems have become the method of choice for transgenic research. The Tet-Off systems have a number of advantages, including robust target induction using a relatively benign effector molecule. However, use of the Tet-On system has been fraught with difficulties, including high background expression in the absence of effector molecules and inconsistent gene induction. Recently, second generation Tet-On transactivators (TAs) have been described. In HeLa cells, they are far more efficient than the original reverse TA protein, and they exhibit lower background activity in the absence of effectors. Here we examine the most promising TA in transgenic Drosophila and characterize its in vivo properties. We report that low levels of doxycycline, when added to normal fly food, efficiently and rapidly induce target transgenes in adults, larvae, and embryos. This TA is superior to all other Tet-On proteins, and its performance is comparable to that of the widely used Tet-Off TA. In addition, combining the improved Tet-On TA with the Gal4-UAS (upstream-activating sequence) system produces robust, spatially restricted, temporally controlled transgene induction. Because this Tet-On TA is significantly more efficient than previous ones used in Drosophila, it is also possible to modulate gene induction by controlling the dosage of the antibiotic in the food.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras , Tetraciclina/metabolismo , Activación Transcripcional , Animales , Animales Modificados Genéticamente , Proteínas Bacterianas/genética , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Drosophila melanogaster/genética , CinéticaRESUMEN
Natural Tet repressor (TetR) variants are alpha-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the alpha8-alpha9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 10(5) M(-1) for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 x 10(9) M(-1) and 8.0 x 10(9) M(-1), about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length-to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction.
Asunto(s)
Proteínas Represoras/química , Proteínas Represoras/genética , Tetraciclinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia Conservada/genética , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Isofunctional tetracycline repressor (TetR) proteins isolated from different bacteria show a sequence identity between 38 and 88% of the residues. Their active state is a homodimer formed by a four-alpha-helix bundle as the main interaction motif. We utilize this sequence variation of isofunctional proteins to determine residues contributing to the stability of the four-helix bundle. The thermodynamic stabilities of two TetR proteins with 63% sequence identity were determined by urea-induced reversible denaturation followed by fluorescence and circular dichroism. Both methods yield identical results. The deltaG(o)U (H2O) values are 60 and 75 kJ x mol(-1). We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ x mol(-1) stability difference. Successive size reduction of the exchanged portion yielded two single residues affecting the overall protein stability. The P184Q exchange leads to a more stable protein, whereas the G181D exchange located at the solvent's exposed edge of the four-helix bundle is solely responsible for the reduced stability. Additional mutants based on crystal structures of TetR do not reveal any hint for steric interference of the Asp181 side chain with neighboring residues. Thus, this is an example for the role played by surface-exposed turn residues for the stability of four-helix bundles. We assume that the larger conformational flexibility of Gly and the reduction of the negative surface charge could favor formation of the turn on the edge of the four-helix bundle.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Represoras/química , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Ácido Aspártico/química , Ácido Aspártico/genética , Proteínas Bacterianas/genética , Simulación por Computador , Dimerización , Escherichia coli/química , Escherichia coli/genética , Vectores Genéticos/síntesis química , Glicina/química , Glicina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Mapeo Peptídico , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Represoras/genética , Tetraciclina/antagonistas & inhibidores , Termodinámica , UreaRESUMEN
Live-attenuated human immunodeficiency virus type 1 (HIV-1) variants have shown great promise as AIDS vaccines, but continued replication can lead to the selection of faster-replicating variants that are pathogenic. We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. These designer "HIV-rtTA" viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells, and the rate of replication can be fine-tuned by simple variation of the dox concentration. These HIV-rtTA viruses provide a tool to perform genetics, e.g., selection and optimization experiments, with the E. coli-derived Tet reagents in a eukaryotic background. Furthermore, such viruses may represent improved vaccine candidates because their replication can be turned on and off at will.
Asunto(s)
Proteínas de Escherichia coli , Productos del Gen tat/genética , VIH-1/genética , VIH-1/fisiología , Receptores de Superficie Celular , Proteínas Represoras/genética , Activación Transcripcional , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Células Quimiorreceptoras , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Regulación Viral de la Expresión Génica , Productos del Gen tat/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Insercional , Proteínas Represoras/metabolismo , Linfocitos T/virología , Transfección , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
A major goal in gene therapy is to develop efficient gene transfer protocols that allow tissue-specific, long-term and tightly regulated expression of the desired transgene. This objective is becoming more attainable through the co-evolution of gene transfer vectors and regulation systems. The ideal vector should efficiently transduce non-dividing cells with minimal toxicity, thus endowing the system with persistent transgene expression. The helper-dependent adenovirus vectors meet these requirements, as demonstrated in various studies in the literature. The most promising regulation system is the tet-on system, which has low basal transcriptional activity and high inducibility. To explore the regulated transgene expression in the context of a helper-dependent vector, we constructed the HD-TET-IFN vector, containing the mIFN(alpha) gene under the control of the tetracycline inducible transactivator rtTA2(s)-S2. Mice injected with HD-TET-IFN showed high levels of serum mIFN(alpha) only upon transcriptional activation. The transgene expression was reinducible to the same high level up to 3 months p.i., and the amount of expressed cytokine could be regulated by dosing doxycycline. Transcriptional activation of mIFN(alpha) induced by doxycycline resulted in prolonged survival and reduced liver damage in HD-TET-IFN-injected mice challenged with a lethal dose of coronavirus. Activation of antiviral genes mediated by doxycycline-dependent mIFN(alpha) expression was also observed at low HD-TET-IFN doses. The possibility of controlling gene expression by the combination of HD vectors and the latest tet-on transactivator also holds promise for studying gene function in other animal models.
Asunto(s)
Adenoviridae/genética , Antibacterianos/uso terapéutico , Doxiciclina/uso terapéutico , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Interferón-alfa/genética , Alanina Transaminasa/sangre , Animales , Carcinoma Hepatocelular , Línea Celular , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Expresión Génica , Regulación de la Expresión Génica , Hepatitis Viral Animal/terapia , Humanos , Interferón-alfa/análisis , Interferón-alfa/sangre , Hígado/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Animales , ARN Mensajero/análisis , Transactivadores/genética , TransgenesRESUMEN
We have determined the role played by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in carbon catabolite repression (CCR) of xylose utilization in Bacillus megaterium. For that purpose we have cloned, sequenced and inactivated the genes ptsH and ptsl of B. megaterium, encoding HPr and EI of the PTS, respectively. While glucose uptake of a ptsHI mutant is not affected at 12.5 mM of glucose, CCR of the xyl operon is reduced in this mutant from 16-fold to 3-fold. This may be attributed to the loss of the corepressor of CcpA, HPr(Ser-P), or could result from the slower growth rate of the mutant. In contrast, CCR exerted by fructose or mannitol is completely abolished. We conclude that glucose triggers additional mechanisms of CCR than fructose or mannitol. The remaining 3-fold glucose repression is relieved in a strain in which ptsHI and glk, encoding glucokinase, are inactivated. This result indicates that glucose metabolism is necessary for CCR. The ability of the ptsHI mutant to take up glucose suggests the existence of a second, non-PTS glucose uptake system. The Km and vmax values of this transporter ranged between 2 and 5 mM and 154 to 219 nmol/[(mg protein)*min], respectively.
Asunto(s)
Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas Bacterianas , Fructosa/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Bacillus megaterium/crecimiento & desarrollo , Bacillus subtilis/genética , Transporte Biológico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Marcadores Genéticos , Cinética , Manitol/metabolismo , Datos de Secuencia Molecular , Operón , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
The gram-positive bacterium Bacillus subtilisis capable of using numerous carbohydrates as single sources of carbon and energy. In this review, we discuss the mechanisms of carbon catabolism and its regulation. Like many other bacteria, B. subtilis uses glucose as the most preferred source of carbon and energy. Expression of genes involved in catabolism of many other substrates depends on their presence (induction) and the absence of carbon sources that can be well metabolized (catabolite repression). Induction is achieved by different mechanisms, with antitermination apparently more common in B. subtilis than in other bacteria. Catabolite repression is regulated in a completely different way than in enteric bacteria. The components mediating carbon catabolite repression in B. subtilis are also found in many other gram-positive bacteria of low GC content.
Asunto(s)
Bacillus/genética , Bacillus/metabolismo , Metabolismo de los Hidratos de Carbono , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Inducción Enzimática , Represión Enzimática , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Operón , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismoRESUMEN
We have examined anhydrotetracycline (atc) binding to Tet repressor (TetR) in dependence of the Mg(2+) concentration. Of all tc compounds tested so far, atc has the highest affinity for TetR, with a K(A) of 9.8 x 10(11) M(-1) in the presence of Mg(2+) and 6.5 x 10(7) M(-1) without Mg(2+). Thus, it binds TetR with 500-fold higher affinity than tc under both conditions. The Mg(2+)-free binding of atc to TetR leads to induction in vitro, demonstrating that the metal is not necessary to trigger the associated conformational change. To obtain more detailed information about Mg(2+)-free induction, we constructed and prepared to homogeneity four single-alanine substitution mutants of TetR. Three of them affect residues involved in contacting Mg(2+) (TetR H100A, E147A, and T103A), and one altered residue contacts tc TetR N82A. TetR H100A and E147A are induced by atc, with and without Mg(2+), showing 110-fold and 1000-fold decreased Mg(2+)-dependent and unchanged Mg(2+)-independent atc binding, respectively. Thus, the contacts of these residues to Mg(2+) are not necessary for induction. TetR N82A is not inducible under any of the conditions employed and shows an about 4000-fold decreased atc binding constant. The Mg(2+)-dependent affinity of TetR T103A for atc is only 400-fold reduced, but no induction with atc was observed. Thus, Thr103 must be essential for the conformational change associated with induction in the absence of Mg(2+).
Asunto(s)
Proteínas Bacterianas/biosíntesis , Magnesio/química , Proteínas Represoras/biosíntesis , Alanina/química , Alanina/genética , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Escherichia coli , Sustancias Macromoleculares , Magnesio/metabolismo , Modelos Químicos , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Espectrometría de Fluorescencia , Tetraciclinas/biosíntesis , Tetraciclinas/metabolismo , VolumetríaRESUMEN
The excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms. The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die. The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome. The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon. When Tc diffuses into the cell it chelates Mg(2+) and the complex [MgTc](+) binds to (TetR)(2) to form the induced complex (TetR small middle dot[MgTc](+))(2). This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc. Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two [MgTc](+) to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dot[MgTc](+))(2) is no longer able to recognize and bind to tetO. Since the switching mechanisms of the TetR/[MgTc](+) system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.
RESUMEN
We have analyzed the tryptophan (trp) fluorescence-decay kinetics of single trp mutants of the Tet repressor protein in the free, the tet operator and anhydrotetracycline (atc)-bound states. The position of the single trp varies between residues 164 and 171, in close proximity to one entrance of the tetracycline-binding pocket. A good fit of the trp fluorescence decay needed generally three exponentials. The decay times vary with detection wavelength, the extent of this variation being correlated to the variation of the emission maximum. Quenching experiments with neutral (acrylamide), cationic (N-methylpyridinium chloride) and anionic quencher (KI) support the interpretation of the three fluorescence components within a conformer model. Operator and atc binding change the ratio of the relative amplitudes of the medium- and long-lived component, thus pointing to structural changes as indicated also by the changes in decay time. Since the fluorescence decay is different between the free, atc- and operator-bound states we conclude that the protein structure is different in each of these three states. The fluorescence quenching constants reflect not only the variation in solvent exposure with position, but also the fact that the net surface charge in this region is negative, because the quenching constants by the cationic quencher are up to 10-fold higher. The atc fluorescence appears to decay monoexponentially with about the same decay time for all mutants, except W170, in which the trp residue sterically interferes with atc.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Represoras/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Regiones Operadoras Genéticas , Conformación Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Espectrometría de Fluorescencia , Tetraciclinas/metabolismo , Triptófano/químicaRESUMEN
Effector triggered conformational changes of proteins such as regulators of transcription, receptors, or enzymes are the molecular basis for regulation in biology. Most proteins perform their biological functions intracellularly, in the presence of many potential interaction partners. Studies of conformational changes have mainly been performed in vitro using sophisticated physical and biochemical methods that usually require purified proteins. Here we describe the observation of conformational changes of Tet repressor in the cytoplasm of growing Escherichia coli cells, analyzed by ligand dependent disulfide crosslinking of cysteine residues substituted into mobile regions of the protein. The amount of protein undergoing the structural change is quantitatively linked to the concomitant induction of transcription of a reporter gene.
Asunto(s)
Escherichia coli/citología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Cisteína/genética , Cisteína/metabolismo , Citoplasma/química , Citoplasma/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Disulfuros/metabolismo , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/genética , Genes Reporteros/genética , Ligandos , Modelos Moleculares , Mutación/genética , Regiones Operadoras Genéticas/genética , Oxidación-Reducción , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Tetraciclina/metabolismo , Tetraciclina/farmacologíaRESUMEN
Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P(tet) promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2(S)-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.
Asunto(s)
Proteínas Portadoras , Mutación , Proteínas Represoras/genética , Tetraciclinas/farmacología , Transactivadores/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Doxiciclina/farmacología , Células HeLa , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
The tc-responsive TetR protein allows the investigation of various transcriptional activators in respective fusion proteins. We have fused eight well-known human activator domains to the C-terminus of TetR and determined the properties of the resulting transactivators using a tetracycline-responsive promoter in three human cell lines (HeLa, BJAB, and Jurkat). Several-hundred-fold activation was exclusively obtained with the acidic p65 domain from NF-kappaB and with VP16, which served as a positive control. In contrast, at least 10-fold lower factors of activation were achieved with ITF-1, ITF-2, and MTF-1. The induction properties of the p65 domain are identical to those of VP16 in all three human cell lines and when fused to the reverse TetR. The combination of the novel reverse p65 fusion with the TetR(B/E)-KRAB construct resulted in active silencing and full activation. This is the first report of an expression system with minimal basal activity and high induction levels without viral protein domains.
Asunto(s)
FN-kappa B/genética , Tetraciclina/farmacología , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Sitios de Unión/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Resistencia a la Tetraciclina/genética , Transactivadores , Factor de Transcripción ReIA , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The tetracycline repressor (TetR) regulates the most abundant resistance mechanism against the antibiotic tetracycline in grain-negative bacteria. The TetR protein and its mutants are commonly used as control elements to regulate gene expression in higher eukaryotes. We present the crystal structure of the TetR homodimer in complex with its palindromic DNA operator at 2.5 A resolution. Comparison to the structure of TetR in complex with the inducer tetracycline-Mg2+ allows the mechanism of induction to be deduced. Inducer binding in the repressor core initiates conformational changes starting with C-terminal unwinding and shifting of the short helix a6 in each monomer. This forces a pendulum-like motion of helix a4, which increases the separation of the attached DNA binding domains by 3 A, abolishing the affinity of TetR for its operator DNA.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Oligodesoxirribonucleótidos/metabolismo , Regiones Operadoras Genéticas/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Tetraciclina/farmacología , Regulación Alostérica/efectos de los fármacos , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Escherichia coli/química , Escherichia coli/genética , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Conformación Proteica/efectos de los fármacos , Proteínas Represoras/genética , Homología de Secuencia , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por Sustrato , Tetraciclina/química , Tetraciclina/metabolismoRESUMEN
A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.