RESUMEN
Metastatic differentiated thyroid cancers (DTC) are resistant to traditional chemotherapy. Kinase inhibitors have shown promise in patients with progressive DTC, but dose-limiting toxicity is commonplace. HSP90 regulates protein degradation of several growth-mediating kinases such as RET, and we hypothesized that HSP90 inhibitor (AUY922) could inhibit RET-mediated medullary thyroid cancer (MTC) as well as papillary thyroid cancer (PTC) cell growth and also radioactive iodine uptake by PTC cells. Studies utilized MTC cell lines TT (C634W) and MZ-CRC-1 (M918T) and the PTC cell line TPC-1 (RET/PTC1). Cell viability was assessed with MTS assays and apoptosis by flow cytometry. Signaling target expression was determined by western blot and radioiodine uptake measured with a gamma counter. Prolonged treatment of both MTC cell lines with AUY922 simultaneously inhibited both MAPK and mTOR pathways and significantly induced apoptosis (58.7 and 78.7% reduction in MZ-CRC-1 and TT live cells respectively, following 1âµM AUY922; P<0.02). Similarly in the PTC cell line, growth and signaling targets were inhibited, and also a 2.84-fold increase in radioiodine uptake was observed following AUY922 administration (P=0.015). AUY922 demonstrates in vitro activity against MTC and PTC cell lines. We observed a potent dose-dependent increase in apoptosis in MTC cell lines following drug administration confirming its anti-tumorigenic effects. Western blots confirm inhibition of pro-survival proteins including AKT suggesting this as the mechanism of cell death. In a functional study, we observed an increase in radioiodine uptake in the PTC cell line following AUY922 treatment. We believe HSP90 inhibition could be a viable alternative for treatment of RET-driven chemo-resistant thyroid cancers.
RESUMEN
Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from 'stand-by' to 'P-decoding' site; (iii) dissociation of 30S-poly(U)-NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain-30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S-IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem-loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.
Asunto(s)
Factores de Iniciación de Péptidos/fisiología , Escherichia coli/genética , Factor 3 de Iniciación Eucariótica , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero , ARN de Transferencia de Metionina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Ribosomas/metabolismoRESUMEN
Two Csp proteins (CspA and CspD) were fused to the green fluorescent protein GFP and expressed from their natural promoters or from an inducible promoter. Fluorescence microscopy and computerized image analysis indicate that in Escherichia coli growing at 37 degrees C CspD localizes in the nucleoid like the control H-NS while CspA occupies a polar position away from the nucleoid. Following cold shock CspA maintains its location, while CspD is not sufficiently expressed to permit its localization. The different localization of CspA and CspD indicates that these proteins play different roles in the cell in spite of their extensive structural similarity.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Factores de Transcripción/biosíntesis , Proteínas Bacterianas/análisis , Western Blotting , Nucléolo Celular , Frío , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Microscopía Fluorescente , Factores de Transcripción/análisisRESUMEN
Gastrodia elata Bl. (GE) is a traditional Chinese herb that is commonly used in Chinese communities to treat convulsive disorders such as epilepsy. The purpose of the present study was to determine the anticonvulsive and free radical activities of GE in rats. In vitro studies were conducted by using brain tissue from 6 male Sprague-Dawley (SD) rats treated with 120 microg/ml of kainic acid (KA), with or without the addition of various concentrations of GE. In vivo studies were conducted in a total of 30 male SD rats divided into 5 groups of 6 rats which were treated as follows: 1) the normal group received an intraperitoneal injection (i.p.) of PBS (Phosphate buffer saline, 1 ml/kg); 2) the control group received KA (12 mg/kg) i.p.; 3) the GE 1.0 group received oral administration of GE 1.0 g/kg 30 min prior to KA administration; 4) the GE 0.5 group received oral administration of GE 0.5 g/kg 30 min prior to KA administration; 5) the PH group received oral administration of phenytoin 20 mg/kg 30 min prior to KA administration. Seizures were verified by behavioral observations, electroencephalograph (EEG) and electromyography (EMG). Lipid peroxide levels in the rat brain, luminol chemiluminescence (CL) and lucigenin-CL in the peripheral blood were measured simultaneously after behavioral observations. The results indicate that GE administration significantly reduced KA-induced lipid peroxide levels in vitro. Oral administration of GE 1.0 g/kg and phenytoin 20 mg/kg significantly reduced counts of wet dog shakes (WDS), paw tremor (PT) and facial myoclonia (FM) in KA-treated rats. In addition, oral administration of GE 1.0 g/kg significantly delayed the onset of WDS, from 30 min in the control group to 46 min in the 0.5 g/kg group, and 63 min in the GE 1.0 g/kg group. A significantly reduced level of lipid peroxides in the rat brain was found in the GE 1.0 g/kg, 0.5 g/kg, and phenytoin 20 mg/kg groups. The GE 1.0 g/kg group showed significant reduction of luminol-CL and lucigenin-CL counts in the peripheral blood compared to the control group. The results of the present study demonstrate that GE has anticonvulsive and free radical scavenging activities. Further studies are needed to determine the clinical effectiveness of GE as an anticonvulsant in humans.
Asunto(s)
Anticonvulsivantes/farmacología , Encéfalo/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Epilepsia/prevención & control , Depuradores de Radicales Libres/farmacología , Peroxidación de Lípido/efectos de los fármacos , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Electroencefalografía , Electromiografía , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Epilepsia/fisiopatología , Agonistas de Aminoácidos Excitadores , Técnicas In Vitro , Ácido Kaínico , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The promoter of hns, the structural gene for the abundant nucleoid-associated protein H-NS of Escherichia coli, contains, downstream of the initiation site, two four bp-long 'CG clamps', one of which overlaps the potential target sequence (CCAAT) of CspA, the cold-shock transcriptional enhancer of this gene. To establish the role of these potential regulatory signals during the cold-shock activation of hns, the CCCCAAT sequence has been subjected to mutagenesis, weakening the strength of the CG clamp and scrambling or inverting the CCAAT sequence. The resulting mutated hns promoters were placed in front of a reporter gene (cat) and their activity was studied in cells subjected to cold-shock under conditions where the increase in the concentration of CspA is either large or small. Our results allow us to conclude that although not essential, the CCCCAAT sequence, mainly due to the presence of the CG clamp, may play an important role in the CspA-mediated regulation of hns expression at both transcriptional and translational levels.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Mutagénesis/genética , Regiones Promotoras Genéticas , Transcripción Genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Frío , Cartilla de ADN/química , Electroforesis en Gel de Agar , Eliminación de Gen , Biosíntesis de Proteínas , ARN Mensajero/genéticaAsunto(s)
Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Secuencia de Aminoácidos , Bacterias/genética , Factor 1 Eucariótico de Iniciación/genética , Factor 1 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.
Asunto(s)
Adaptación Fisiológica/genética , Emparejamiento Base/genética , Frío , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Mensajero/química , ARN Ribosómico 16S/química , Secuencias Reguladoras de Ácidos Nucleicos/genética , Aldehídos/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Butanonas , División Celular , Dimetilsulfóxido/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Genes Reporteros/genética , Mutación/genética , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/química , Ribosomas/metabolismoRESUMEN
The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.
Asunto(s)
Geobacillus stearothermophilus/química , Factores de Iniciación de Péptidos/química , Biosíntesis de Proteínas , ARN de Transferencia de Metionina/química , Sitios de Unión , Geobacillus stearothermophilus/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , N-Formilmetionina/química , N-Formilmetionina/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factor 2 Procariótico de Iniciación , Conformación Proteica , ARN de Transferencia de Metionina/metabolismo , TermodinámicaRESUMEN
Vanillyl alcohol (VA) is a component of Gastrodia elata Bl. (GE), which is a traditional Chinese herb widely used to treat convulsive disorders or dizziness. This study examined the role of VA in the anticonvulsive properties of GE in a Sprague-Dawley rat model of epilepsy. The anticonvulsive and free radical scavenging activities of VA were examined after intracortical injection of ferric chloride (100 mM, 8 microl) to induce epileptic seizures. These seizures were verified by behavioral observations and electroencephalographic (EEG) and electromyographic (EMG) recordings. Ferric chloride injection resulted in increased lipid peroxide levels in the ipsilateral and contralateral cerebral cortex, and increased luminol-chemiluminescence (CL) and lucigenin-CL counts in the peripheral blood. Intraperitoneal injection (i.p.) of VA (200 mg/kg or 100 mg/kg) or phenytoin 10 mg/kg prior to ferric chloride administration significantly inhibited wet dog shakes (WDS) and lipid peroxide levels in the bilateral cerebral cortex. VA 200 mg/kg also significantly reduced luminol-CL and lucigenin-CL counts in the peripheral blood, but no significant effect was observed following administration of VA 100 mg/kg or phenytoin. These data indicate that VA has both anticonvulsive and suppressive effects on seizures and lipid peroxidation induced by ferric chloride in rats. Data from the present study also demonstrate that VA has free radical scavenging activities, which may be responsible for its anticonvulsive propertics. This finding is consistent with the results from previous studies that generation of superoxide radical evoked by injection of iron salt into rat brain plays a critical role in ferric chloride-induced seizures. In addition, the results of the present study suggest that the anticonvulsive effect of GE may be attributable, at least in part, to its VA component.
Asunto(s)
Anticonvulsivantes/farmacología , Alcoholes Bencílicos/farmacología , Epilepsia/tratamiento farmacológico , Depuradores de Radicales Libres/farmacología , Acridinas , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cloruros , Convulsivantes/antagonistas & inhibidores , Convulsivantes/toxicidad , Medicamentos Herbarios Chinos/farmacología , Electroencefalografía , Electromiografía , Epilepsia/sangre , Epilepsia/inducido químicamente , Compuestos Férricos/antagonistas & inhibidores , Compuestos Férricos/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Peróxidos Lipídicos/sangre , Peróxidos Lipídicos/metabolismo , Mediciones Luminiscentes , Luminol , Masculino , Plantas Medicinales , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Analytical ultracentrifugation studies indicated that the C-terminal domains of IF2 comprising amino acid residues 520-741 (IF2 C) and 632-741 (IF2 C-2) bind fMet-tRNA with similar affinities (K(d) at 25 degrees C equal to 0.27 and 0.23 microM, respectively). Complex formation between fMet-tRNA(fMet) and IF2 C or IF2 C-2 is accompanied by barely detectable spectral changes as demonstrated by a comparison of the Raman spectra of the complexes with the calculated sum of the spectra of the individual components. These results and the temperature dependence of the K(d) of the protein-RNA complexes indicate that complex formation is not accompanied by obvious conformational changes of the components, and possibly depends on a rather small binding site comprising only a few interacting residues of both components.
Asunto(s)
Geobacillus stearothermophilus , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , ARN de Transferencia de Metionina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Peso Molecular , Conformación de Ácido Nucleico , Factor 2 Procariótico de Iniciación , Unión Proteica , Conformación Proteica , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Transferencia de Metionina/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Espectrometría Raman , Temperatura , Termodinámica , UltracentrifugaciónRESUMEN
Previous protein unfolding studies had suggested that IF2 C, the 24. 5-kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains. In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule. Comparison of the circular dichroism and Trp fluorescence changes induced by increasing concentrations of guanidine hydrochloride demonstrated that IF2 C indeed consists of two subdomains: the more stable N-terminal (IF2 C-1) subdomain containing Trp-599, and the less stable C-terminal (IF2 C-2) subdomain containing Trp-721. Isolated subdomain IF2 C-2, which consists of just 110 amino acids (from Glu-632 to Ala-741), was found to bind fMet-tRNA with the same specificity and affinity as native IF2 or IF2 C-domain. Trimming IF2 C-2 from both N and C termini demonstrated that the minimal fragment still capable of fMet-binding consists of 90 amino acids. IF2 C-2 was further characterized by circular dichroism; by urea-, guanidine hydrochloride-, and temperature-induced unfolding; and by differential scanning calorimetry. The results indicate that IF2 C-2 is a globular molecule containing predominantly beta structures (25% antiparallel and 8% parallel beta strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , ARN de Transferencia de Metionina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Rastreo Diferencial de Calorimetría , Cartilla de ADN , Guanidina , Calor , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/genética , Factor 2 Procariótico de Iniciación , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calorimetría , Cationes Monovalentes/farmacología , Cromatografía en Gel , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
This study investigated the anticonvulsant effect of Uncaria rhynchophylla (UR) and the physiological mechanisms of its action in rats. A total of 70 male Sprague-Dawley (SD) rats were selected for study. Thirty four of these rats were divided into 5 groups as follows: 1) CONTROL GROUP (n = 6): received intraperitoneal injection (i.p.) of kainic acid (KA, 12 mg/kg); 2) UR1000 group (n = 10), 3) UR500 group (n = 6) 4) UR250 group, received UR 1000, 500, 250 mg/kg i.p. 30 min prior to KA administration, respectively; 5) Contrast group: received carbamazepine 20 mg/kg i.p. 30 min prior to KA administration. Behavior and EEG were monitored from 15 min prior to drug administration to 3 hours after KA administration. The number of wet dog shakes were counted at 10 min intervals throughout the experimental course. The remaining 36 rats were used to measure the lipid peroxide level in the cerebral cortex one hour after KA administration. These rats were divided into 6 groups of 6 rats as follows: 1) Normal group: no treatment was given; 2) CONTROL GROUP: received KA (12 mg/kg) i.p.; 3) UR1000 group, 4) UR500 group, 5) UR250 group, received UR 1000, 500, 250 mg/kg i.p. 30 min prior to KA administration, respectively; 6) Contrast group: received carbamazepine 20 mg/kg i.p. 30 min prior to KA administration. Our results indicated that both UR 1000 and 500 mg/kg decreased the incidence of KA-induced wet dog shakes, no similar effect was observed in the UR 250 mg/kg and carbamazepine 20 mg/kg group. Treatment with UR 1000 mg/kg, 500 mg/kg, or 250 mg/kg and carbamazepine 20 mg/kg decreased KA-induced lipid peroxide level in the cerebral cortex and was dose-dependent. These findings suggest that the anticonvulsant effect of UR possibly results from its suppressive effect on lipid peroxidation in the brain.
Asunto(s)
Anticonvulsivantes/farmacología , Ácido Kaínico , Plantas Medicinales/química , Convulsiones/prevención & control , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroencefalografía/efectos de los fármacos , Electromiografía/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Medicina Tradicional China , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamenteRESUMEN
The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA).
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Cloranfenicol O-Acetiltransferasa/genética , Frío , Medios de Cultivo , Cartilla de ADN , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Retroalimentación , Cinética , Sondas de Oligonucleótidos , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
A shift in growth temperature from 37 degrees C to 15 degrees C leads to a dramatic increase in the level of CspA, the major cold shock protein of Escherichia coli. To investigate the molecular basis of this induction, we considered the relevance of transcriptional and posttranscriptional controls by analyzing the steady-state levels of transcripts and the expression of reporter genes in cells carrying a set of cspA promoter fragments of variable length fused to lacZ or cat genes. We demonstrate that: (i) the core cspA promoter (from -40 to +16) responds to cold shock and a mutation at -36 increases the relative activity of the promoter at low temperature by threefold; (ii) the sequences upstream of -40 have a positive effect on expression at 37 degrees C, but no effect on the cold shock response; (iii) by virtue of their influence on mRNA stability, the downstream sequences (from +81 to +165) reduce expression at 37 degrees C and increase the intensity of the cold shock response; (iv) mutations in the GCACATCA and CCAAT motifs, present at +1/-4 and between the -10 and -35 elements, respectively, do not affect the cold shock response of the cspA promoter; (v) following cold shock, a modification of the protein synthetic machinery takes place that allows preferential translation of cspA mRNA relative to the non-cold shock cat and lacZ mRNAs. The quantitatively modest transcriptional activation shown by the core promoter of cspA following cold shock suggests that transcriptional activation can significantly contribute to cold shock induction only when coupled to posttranscriptional controls, such as alterations in mRNA stability and the translational apparatus.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Adaptación Fisiológica , Frío , Mutagénesis , Procesamiento Proteico-PostraduccionalRESUMEN
Purpuromycin, an antibiotic produced by Actinoplanes ianthinogenes, had been reported previously to inhibit protein synthesis. In the present report, we demonstrate that the mechanism of action of this antibiotic is quite novel in that it binds with fairly high affinity to all tRNAs, inhibiting their acceptor capacity. Although more than one molecule of purpuromycin is bound to each tRNA molecule, the inhibitory activity of this antibiotic was found to be selective for the tRNA acceptor function; in fact, after the aminoacylation step, purpuromycin was found to affect none of the other tested functions of tRNA (interaction with the ribosomal P- and A-sites and interaction with translation factors). Accordingly, purpuromycin was found to inhibit protein synthesis only when translation depended on the aminoacylation of tRNA and not when the system was supplemented with pre-formed aminoacyl-tRNAs. Because purpuromycin did not interfere with the ATP-PPi exchange reaction of the synthetase or with the initial interaction of the enzyme with its tRNA substrate, the basis for the inhibition of aminoacylation is presumably the formation of a nonproductive synthetase-tRNA complex in the presence of purpuromycin in which the tRNA is unable to be charged with the corresponding amino acid.
Asunto(s)
ARN de Transferencia/efectos de los fármacos , ARN de Transferencia/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Naftoquinonas/metabolismo , Naftoquinonas/farmacología , Biosíntesis de Proteínas , ARN Bacteriano/efectos de los fármacos , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/genética , ARN de Transferencia de Fenilalanina/efectos de los fármacos , ARN de Transferencia de Fenilalanina/genética , ARN de Transferencia de Fenilalanina/metabolismoRESUMEN
Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time. DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Huella de ADN , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from -282 to +25 with two sites, closely flanking the DNA bend located at -150 from the transcriptional startpoint, partly overlapping the H-NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H-NS and the hns promoter region were studied by examining the effects of FIS and H-NS on in vitro transcription of hns-cat fusions, as well as looking at the effect of FIS on preformed complexes containing H-NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H-NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Secuencia de Bases , Sitios de Unión , ADN Bacteriano , Escherichia coli/metabolismo , Factor Proteico para Inverción de Estimulación , Factores de Integración del Huésped , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
The requirements for the adjustment of fMet-tRNA in the ribosomal P-site have been analyzed by studying the formation of fMet-puromycin in a Bacillus stearothermophilus system. The binding of fMet-tRNA to the 30 S ribosomal subunit is not drastically affected by the omission of GTP, mRNA, mRNA and GTP, or by replacing GTP with GTP analogues. The adjustment of fMet-tRNA in the P site has stricter requirements and fMet-puromycin formation occurred at its maximum rate and extent when fMet-tRNA was bound to 30 S subunits programmed with the AUG triplet or with an mRNA in the presence of GTP. Neither GTP nor the mRNA, however, were found to be essential. Omission of GTP caused only a slight reduction in the rate of fMet-puromycin formation without a significant change of the activation energy, while omission of the template resulted in a requirement for a higher activation energy. In the absence of both GTP and template, however, essentially no fMet-puromycin was formed, indicating that these components cooperate in the adjustment of the initiator tRNA in the P-site. The contribution of various structural elements of the mRNA in determining this adjustment was investigated. It was found that the codon-anticodon interaction and the filling of the ribosomal mRNA channel with a polyribonucleotide are necessary (but not sufficient singly) for the correct orientation of the initiator tRNA in the absence of GTP. The nature of the initiation triplet and the occurrence and/or the strength of the Shine-Dalgarno interaction were also found to contribute to the orientation of the bound fMet-tRNA.
Asunto(s)
Geobacillus stearothermophilus/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN de Transferencia de Metionina/metabolismo , Ribosomas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Codón Iniciador/genética , Escherichia coli/química , Geobacillus stearothermophilus/genética , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Magnesio/farmacología , Factor Tu de Elongación Peptídica/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Poli U/metabolismo , Polirribonucleótidos/metabolismo , Factor 2 Procariótico de Iniciación , Puromicina/análogos & derivados , Puromicina/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/genética , TemperaturaRESUMEN
The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible lambda PL promoter. After induction of transcription by thermal inactivation of the lambda ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10 degrees C, but poor expression was obtained if the cells were incubated at 37 degrees C or 30 degrees C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37 degrees C compared to 10 degrees C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10 degrees C compared to 37 degrees C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37 degrees C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.