RESUMEN
The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF. Because MazE is labile, inhibition of mazE expression results in cell death. We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1vir). In several E. coli strains, we showed that the Delta mazEF derivative strains produced significantly more phages than did the parent strain. In addition, upon induction of K38(P1CM c1ts), nearly all of the Delta mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis. However, most of these cells did not remain viable. Thus, while the Delta mazEF cells die as a result of the lytic action of the phage, most of the mazEF+ cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself. Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to Delta mazEF but not for mazEF+ cultures. Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population. These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms.
Asunto(s)
Bacteriófago P1/patogenicidad , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/virología , Genes Bacterianos , Apoptosis/genética , Bacteriófago P1/fisiología , Endorribonucleasas , Escherichia coli/citología , Lisogenia , Profagos/patogenicidad , Activación Viral , Replicación ViralRESUMEN
The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading. Interesting alternative relates to the function of the UGA codon. Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide. UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure. Here we discuss our investigation on the E. coli SECIS. This includes the follows: 1) The nature of the minimal E. coli SECIS. We found that in E. coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA [34]; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation [34] because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency [35]. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Finally, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Selenocisteína/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Codón/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismoRESUMEN
The discovery of toxin-antitoxin gene pairs (also called addiction modules) on extrachromosomal elements of Escherichia coli, and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures. We have reported on the E. coli mazEF system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and MazE is a labile antitoxin. Here we show that cell death mediated by the E. coli mazEF module can be triggered by several antibiotics (rifampicin, chloramphenicol, and spectinomycin) that are general inhibitors of transcription and/or translation. These antibiotics inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes cell death. Our results have implications for the possible mode(s) of action of this group of antibiotics.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Antitoxinas/genética , Antitoxinas/metabolismo , Apoptosis , Proteínas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
"Addiction modules" consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The Escherichia coli chromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E. coli mazEF system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited. Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEF systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteriófago P1/fisiología , Escherichia coli/virología , Proteínas Virales/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Apoptosis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Bacteriófago P1/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Plásmidos/genética , Proteínas Virales/genéticaRESUMEN
The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death. This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE. Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF. We show that the expression of this module is positively regulated by the factor for inversion stimulation. What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c". When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results. We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions.
Asunto(s)
Apoptosis/genética , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Antitoxinas , Secuencia de Bases , Proteínas Portadoras/metabolismo , Endorribonucleasas , Genes Bacterianos , Factores de Integración del Huésped , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Factores de Elongación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Selenocisteína/metabolismo , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Escherichia coli/genética , Formiato Deshidrogenasas/genética , Formiato Deshidrogenasas/metabolismo , Hidrogenasas/genética , Hidrogenasas/metabolismo , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Selenocisteína/genética , Nucleótidos de Uracilo/genéticaRESUMEN
The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein. Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively, and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Here, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.
Asunto(s)
Escherichia coli/genética , Formiato Deshidrogenasas/genética , Hidrogenasas/genética , Complejos Multienzimáticos/genética , Biosíntesis de Proteínas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Selenio/fisiología , Selenocisteína/metabolismo , Arginina/genética , Arginina/metabolismo , Secuencia de Bases , Codón/genética , Escherichia coli/enzimología , Formiato Deshidrogenasas/química , Formiato Deshidrogenasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrogenasas/química , Hidrogenasas/metabolismo , Metaloproteínas/química , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Genéticos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Conformación de Ácido Nucleico , Nucleótidos/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Selenio/deficiencia , Regiones Terminadoras Genéticas/genética , Valina/genética , Valina/metabolismoRESUMEN
In bacteria, programmed cell death is mediated through "addiction modules" consisting of two genes. The product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. Here we extensively review what is known about those modules that are borne by one of a number of Escherichia coli extrachromosomal elements and are responsible for the postsegregational killing effect. We focus on a recently discovered chromosomally borne regulatable addiction module in E. coli that responds to nutritional stress and also on an antideath gene of the E. coli bacteriophage lambda. We consider the relation of these two to programmed cell death and antideath in bacterial cultures. Finally, we discuss the similarities between basic features of programmed cell death and antideath in both prokaryotes and eukaryotes and the possibility that they share a common evolutionary origin.
Asunto(s)
Apoptosis , Bacteriófago lambda/genética , Escherichia coli/citología , Genes Bacterianos , Plásmidos/genética , Apoptosis/genética , Apoptosis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/virología , Genes Virales , Proteínas Virales/genética , Proteínas Virales/fisiologíaRESUMEN
In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.
Asunto(s)
Bacteriófago lambda/genética , Escherichia coli/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Clonación Molecular , Endopeptidasa Clp , Escherichia coli/crecimiento & desarrollo , Escherichia coli/virología , Genotipo , Guanilil Imidodifosfato/metabolismo , Ligasas/genética , Ligasas/metabolismo , Lisogenia , Mutagénesis Insercional , Plásmidos , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine. This UGA decoding process requires a cis -acting mRNA element called 'selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure. In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon. Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli . Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac' Z fusion. We found that the whole stem-loop RNA structure of the E.coli fdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo . Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon. Based on these observations, we present a new model for the minimal E.coli SECIS.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/genética , Selenocisteína/genética , Secuencia de Bases , Codón de Terminación/genética , ADN Bacteriano/genética , Escherichia coli/metabolismo , Formiato Deshidrogenasas/biosíntesis , Formiato Deshidrogenasas/genética , Genes Bacterianos , Hidrogenasas/biosíntesis , Hidrogenasas/genética , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Mensajero/química , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Selenocisteína/metabolismoRESUMEN
BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue. We report here the localization of the phosphorylation site to histidine 208. Localization of the phosphorylated histidine was carried out in two steps. We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate. This allowed the localization of the phosphorylation site to the last 111 residues. Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor. Histidine 208 was the only histidine which failed both tests. The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins. The bgl system represents a new family of sensory systems. Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Factor Xa/metabolismo , Histidina/genética , Histidina/metabolismo , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , Fosforilación , Proteínas de Unión al ARN/genéticaRESUMEN
The essential trace element selenium (Se) is involved in the form of selenocysteine at the active site of several prokaryotic and eukaryotic proteins called selenoproteins. These proteins have recently attracted attention particularly in relation to their application to human health and new characteristics of the genetic code. We have recently described a selenium bioassay based on a recombinant DNA construct in which the expression of the lac' Z gene in Escherichia coli is proportionally and specifically driven by UGA-directed selenocysteine incorporation. Here we have further developed this bioassay for more rapid and sensitive detection and measurement of selenium that permits screening of the selenium status on agar plates. Again, the inclusion of selenium into the lac'Z-fusion product is reflected by the level of beta-galactosidase activity, which in turn is reflected by the intensity of fluorescence on agar plates. This fluorescing agent is a 4-methylumbelliferyl moiety which is released through the cleavage by the enzyme of 4-methylumbelliferyl-beta-D-galactoside. The intensity of the fluorescence is easily detected by uv irradiation and photographed by polaroid or video cameras.
Asunto(s)
Colorantes Fluorescentes , Selenio/análisis , Agar , Bioensayo/métodos , Clonación Molecular , Escherichia coli/genética , Galactósidos/química , Himecromona/análogos & derivados , Himecromona/química , Selenio/sangre , Sensibilidad y Especificidad , beta-Galactosidasa/químicaRESUMEN
"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.
Asunto(s)
Cromosomas Bacterianos , Proteínas de Escherichia coli , Escherichia coli/genética , Guanosina Tetrafosfato/farmacología , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Proteínas de Unión al ADN , Endorribonucleasas , Escherichia coli/citología , Eliminación de Gen , Datos de Secuencia Molecular , Mutación , Regiones Promotoras GenéticasRESUMEN
The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR-lac'Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1-lac'Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3' side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5' and of Escherichia coli 16S rRNA. A model of trpR+1-lac'Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5' end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.
Asunto(s)
Sistema de Lectura Ribosómico , Código Genético , Sistemas de Lectura , Bacteriófago T4/genética , Secuencia de Bases , Escherichia coli/genética , Genes Bacterianos , Genes Virales , Datos de Secuencia MolecularRESUMEN
The trace element selenium (Se) is included in the form of selenocysteine (Sec) at the active site of several prokaryotic and eukaryotic proteins known as selenoproteins (SePro). The growing implications of SePro in cell physiology and human health point to the need for an adequate means of assessing Se status in biological fluids. Here, we describe a new approach based on a recombinant DNA construct, in which the expression of the 'lacZ gene in Escherichia coli is proportionally and specifically driven by UGA-directed Sec incorporation. Se status is determined in samples of rat blood first treated by acid hydrolysis for protein degradation. As compared to other methods, this simple, sensitive bioassay (BIO) for determining Se status seems to be unique in its ability to measure all functional Sec residues in SePro in blood serum.
Asunto(s)
ADN Recombinante , Selenio/sangre , Animales , Secuencia de Bases , Bioensayo , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Oligorribonucleótidos , RatasRESUMEN
The type I iodothyronine 5'-deiodinase (5'-DI) present in rat liver and kidney has recently been demonstrated to be a selenoprotein. The goal of the present study was to examine in detail the effect of selenium (Se) deficiency on 5'-DI at the protein and mRNA levels. In weanling rats fed a selenium-deficient (Se(-)) diet for 6 weeks, 5'-DI activity was decreased 91 and 69% relative to control activities in liver and kidney, respectively. Administration of 3,5,3'-triiodothyronine resulted in a 2-fold increase in 5'-DI activity in control animals, but had little or no effect on 5'-DI activity in Se(-) animals. Western analysis using a specific antiserum directed against a bacterial fusion protein containing the carboxyl-terminal half of the 5'-DI protein demonstrated that this decrease in 5'-DI activity in Se(-) animals was explained by a marked decrease in 5'-DI protein. Administration of Se to Se(-) animals resulted in parallel increases in 5'-DI protein and activity over a 72-h time period. It was also shown that selenium deficiency was accompanied by a 40% decrease in 5'-DI mRNA levels in the kidney, but not in the liver. In both tissues, the administration of 3,5,3'-triiodothyronine resulted in increased 5'-DI mRNA levels which were not altered by selenium status. These studies indicate that selenium deficiency decreases 5'-DI activity by decreasing the amount of 5'-DI protein. The mechanism of this impairment in enzyme synthesis appears to be a defect in translation, presumably due to a block in the UGA-directed selenocysteine incorporation in selenium deficiency.
Asunto(s)
Hipertiroidismo/metabolismo , Yoduro Peroxidasa/biosíntesis , Riñón/metabolismo , Hígado/metabolismo , Selenio/deficiencia , Animales , Secuencia de Bases , Dieta , Escherichia coli/genética , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/inmunología , Metaloproteínas/biosíntesis , Metaloproteínas/genética , Metaloproteínas/inmunología , Datos de Secuencia Molecular , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/inmunología , Triyodotironina/metabolismo , DesteteRESUMEN
In Escherichia coli a UGA codon can be efficiently suppressed by a suppressor tRNA(Trp) called Su9. Here, we show that the level of UGA suppression is determined by the nature of the nucleotide at the 5' side of the anticodon of the suppressor (position 33). UGA suppression occurs when a pyrimidine residue is located in position 33 of the tRNA, and suppression is more efficient with a U than with a C in this position. On the other hand, when a purine residue is located at this position UGA suppression is extremely low. These results show that in the case of tRNA Su9, the UGA codon context effect does not require base pairing between the nucleotide at the 3' side of the codon and the 5' side of the anticodon.
Asunto(s)
Anticodón/genética , Codón de Terminación/genética , Escherichia coli/genética , ARN de Transferencia de Triptófano/genética , Supresión Genética/genéticaRESUMEN
The trace element selenium has recently attracted attention, particularly because (i) selenocysteine is involved in the active site of various prokaryotic and eukaryotic enzymes, some of which have a role in human health; (ii) selenocysteine incorporation into these proteins is coded by UGA codons; and (iii) as a result, selenocysteine is now considered to be the 21st amino acid in an expanded genetic code. Here, we built recombinant DNA constructs in which expression of the lac'Z gene is driven in Escherichia coli by UGA-directed selenocysteine incorporation. In this system, levels of beta-galactosidase activity are proportionally and specifically related to the presence and concentrations of several specific simple selenium derivatives. The system can thus be used as a sensitive bioassay for their determination. This bioassay is one of a few using recombinant DNA technology to provide a reporter for simple detection of a chemical trace element.
Asunto(s)
Bioensayo/métodos , ADN Recombinante , Selenio/análisis , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Secuencia de Bases , Codón/genética , Escherichia coli/enzimología , Escherichia coli/genética , Estudios de Evaluación como Asunto , Formiato Deshidrogenasas/genética , Genes Bacterianos , Genes Reporteros , Operón Lac , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , Selenocisteína/metabolismoRESUMEN
The genetic code, once thought to be rigid, has been found to be quite flexible, permitting several different reading alternatives. One of these is translational frameshifting, a process programmed in the mRNA sequence and which enables a +1 or -1 shift from the reading frame of the initiation codon. So far, the involvement of translational frameshifting in gene expression has been described mainly in viruses (particularly retroviruses), retrotransposons, and bacterial insertion elements. In this MicroReview, we present a survey of the cellular genes, mostly in Escherichia coli, which have been found to be expressed through a translational frameshifting process, as well as a discussion of the regulatory implications of this process.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos/genética , Biosíntesis de Proteínas/genética , Secuencia de Bases , Codón , Datos de Secuencia MolecularRESUMEN
Protein O of bacteriophage lambda is a short-lived protein which has a key role in the replication of the phage DNA in Escherichia coli. Here we present evidence that lambda O degradation is energy dependent: it is impaired by cyanide and alpha-methylglucoside, both of which inhibit cellular energy metabolism. Removal of these inhibitors restored the degradation of lambda O. Our experiments suggest that limited amounts of cellular energy are sufficient to support lambda O degradation. In addition, degradation of lambda O protein is prevented by a mutation in the E. coli clpP gene, but not by a mutation in the clpA gene. These results suggest that the ClpP protease is involved in the energy-dependent degradation of the lambda O protein.