RESUMO
Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A single nucleotide substitution changing the Shine-Dalgarno box of carA (GGAGG) into the sequence TGAGG reduces translation of carA sevenfold. This result supports the view that extensive complementarity between the Shine-Dalgarno sequence and 16S RNA contributes significantly to the efficiency of translation when the latter starts at a weak initiation triplet.
Assuntos
Códon , Escherichia coli/genética , Óperon Lac , Biossíntese de Proteínas , RNA Mensageiro , Sequência de Bases , Genes Bacterianos , Dados de Sequência Molecular , RNA Bacteriano/genética , RNA Ribossômico 16S/genéticaRESUMO
The 12 genes which in E. coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5' non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the repressor, a hexameric protein. In vitro binding experiments with purified repressor (a gift from W. K. Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAb and the bipolar argECBH cluster. A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only. Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves. Symmetrical contacts in the minor groove with A residues have also been identified. Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes. Although the wild-type operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity. Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box. The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect. However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced. Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate Oc type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed. The significance of this lack of correlation is discussed.
Assuntos
Arginina , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Operadoras Genéticas , Óperon , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Consenso , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Relação Estrutura-AtividadeRESUMO
The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation (carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.
Assuntos
Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Genes Bacterianos , Óperon , Sequência de Bases , Dados de Sequência Molecular , Mutação , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Pirimidinas , Transcrição GênicaRESUMO
The control region of the carAB operon, encoding carbamoylphosphate synthetase, comprises two tandem promoters (P1, upstream and P2, downstream) located 67 base-pairs apart and repressed respectively by pyrimidines and arginine. RNA polymerase and pure arginine repressor bind to the P2 region in mutually exclusive ways. Repressor protects the two adjacent palindromic ARG boxes overlapping P2 against DNase I. Binding of RNA polymerase to P1 is abnormal; the region protected against DNase I is shifted upstream by about 20 nucleotides with respect to the position expected from the transcription startpoint. This pattern is not due to interference with polymerase binding at P2, since it is observed also in the presence of repressor and on an isolated P1 region. Binding of RNA polymerase is relatively weak and heparin-sensitive suggesting that, in vivo, an ancillary factor is required to promote the formation of an open complex. S1 nuclease mapping experiments show that the simultaneous presence of pyrimidines and arginine represses the downstream arginine-specific promoter (P2) more efficiently than arginine alone. This effect is not due to a direct regulatory interaction between pyrimidines and P2, since it is not observed when P1 is inactivated by insertion mutations or partial deletion. It has been shown that transcription initiated at P1 can proceed even when arginine represses P2. We therefore suggest that P2 operator-arginine repressor complex is destabilized by RNA polymerase binding at P1 or transcription from P1. We describe a novel technique to select for expression-down mutants in a lac fusion context.
Assuntos
Proteínas de Bactérias , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Óperon , Sequência de Bases , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Bacterianos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
The carAB operon of the enterics Escherichia coli K-12 and Salmonella typhimurium LT2, encoding the sole carbamoylphosphate synthetase (CPSase) of these organisms, is transcribed from two promoters in tandem, carP1 upstream and carP2 downstream, repressed respectively by pyrimidines and arginine. We present evidence that the pyrH gene product (the hexameric UMP-kinase) directly participates in the pyrimidine-specific control of carP1 activity. Indeed, we have isolated in E. coli a particular type of pyrH mutation (pyrH41) that retains a quasi-normal UMP-kinase activity, but yet is impaired in the pyrimidine-specific repression of the P1 promoter of the carAB operon of E. coli and of S. typhimurium. Moreover, the pyrimidine-dependent inhibition of in vivo Dam methylase modification of adenine -106 upstream of the carP1 promoter is altered in this pyrH mutant. The recessive pyrH41 allele bears a single C-G to A-T transversion that converts alanine 94 into glutamic acid (A94E). Although overexpression of pyrH41 results in UMP-kinase levels far above that of a wild-type strain, pyrimidine-specific repression of the carAB operon is not restored under these conditions. Similarly, overexpression of the UMP-CMP-kinase gene of Dictyostelium discoideum in the pyrH41 mutant does not restore pyrimidine-mediated control of carP1 promoter activity, in spite of the elevated UMP-kinase activity measured in such transformants. These results indicate that besides its catalytic function in the de novo pyrimidine biosynthesis, E. coli UMP-kinase fulfils an additional, but previously unrecognized role in the regulation of the carAB operon. UMP-kinase might function as the real sensor of the internal pyrimidine nucleotide pool and act in concert with the integration host factor (IHF) and aminopeptidase A (PepA alias CarP and XerB) in the elaboration of the complex nucleoprotein structure required for pyrimidine-specific repression of carP1 promoter activity.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Núcleosídeo-Fosfato Quinase/genética , Regiões Promotoras Genéticas/genética , Adenina/química , Proteínas de Bactérias/metabolismo , Metilação de DNA , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Pirimidinas/metabolismo , Salmonella typhimurium/genética , Ativação TranscricionalRESUMO
By measuring the protection against Dam methylase modification of a GATC sequence located 106 bp upstream of the startpoint of promoter P1 in the control region of the carAB operon (encoding carbamoylphosphate synthetase) we have obtained evidence for a direct correlation between the degree of in vivo occupancy of a specific regulatory target site and the repressibility of the P1 promoter by pyrimidine residues. A high uridine nucleotide pool as well as binding of the carP (alias xerB/pepA) gene product and of the integration host factor (IHF) to the carAB control region are prerequisites to observe this in vivo protection. Purified CarP binds in vitro to the carAB control region and protects against DNase I two approximately 25 bp long stretches, one of which is located just downstream of the GATC sequence. Mutations in this site strongly impair the pyrimidine regulation of the P1 promoter and the interference with Dam methylase modification. These processes are also strongly impaired in the absence of integration host factor and in mutants affected in the IHF site located some 200 bp upstream of this Dam methylase modification site. IHF therefore exerts at least part of its antagonistic effects on P1, i.e. increased expression in minimal medium but increased repression in the presence of pyrimidine residues, indirectly by influencing the formation or the stability of a particular protein-DNA complex. Furthermore, we demonstrate that the distance separating the IHF and Dam methylase target sites is crucial for the in vivo protection and for pyrimidine-mediated regulation of the promoter expression. Mutations altering this distance result in severe reductions of the degree of in vivo protection and, concomitantly, of the repressibility by pyrimidine residues of promoter P1 activity in a way indicative of the formation of a complex nucleoprotein structure. Since neither IHF nor CarP require pyrimidine residues to bind to the carAB control region, at least not in vitro, it is tempting to suggest that IHF and CarP-induced bending and looping provide changes in DNA topology that are required for assembling a specific pyrimidine-dependent nucleoprotein complex that modulates P1 activity.
Assuntos
Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Regiões Promotoras Genéticas , Pirimidinas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Adenina/metabolismo , Aminopeptidases/genética , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Glutamil Aminopeptidase , Fatores Hospedeiros de Integração , Metilação , Dados de Sequência Molecular , Mutação , Óperon/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transcrição Gênica/genética , Nucleotídeos de Uracila/genética , Nucleotídeos de Uracila/metabolismoRESUMO
The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli. The S. solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial regulatory proteins of the Lrp/AsnC family and 30.6% and 25.8% with the archaeal homologues of respectively Methanococcus jannaschii and Pyrococcus furiosus. Transcription initiation from the strong S. solfataricus lrp promoter was analyzed by primer extension mapping. The abundance of the S. solfataricus lrp messenger strongly suggests that this protein might function in archaea as a global transcriptional regulator and genome organizer, as proposed for E. coli Lrp, rather than as a local, specific regulatory protein. Our findings suggest the presence of a eubacterial type of regulatory mechanism in archaea, a situation that is noteworthy indeed, since the transcriptional machinery of archaea is more closely related to that of eukaryotes, whereas these latter apparently do not possess a homologue of Lrp.
Assuntos
Proteínas Arqueais/genética , Genes Arqueais , Leucina , Sulfolobus/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Arqueal , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Binding of integration host factor to its target site, centered around nucleotide -305 upstream of the transcription startpoint, exerts antagonistic effects on the expression of P1, the upstream pyrimidine specific promoter of the E coli and S typhimurium carAB operons. IHF stimulates P1 promoter activity in minimal medium, but also increases the repressibility of this promoter by pyrimidines. We present evidence strongly suggesting that IHF exerts these effects by modulating the binding of another pyrimidine specific regulatory molecule, probably the product of gene carP. The carAB control region contains a GATC Dam methylation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation. This protection requires at least the regulatory carP gene product and a high pyrimidine nucleotide pool and, as shown here, the integration host factor. Whether CarP directly binds to this site or exerts its protective effect indirectly is not yet known. In the absence of IHF (himA) or in mutants affected in the IHF target site this protection is strongly impaired, suggesting that IHF positively influences the formation or the stability of the protective protein-DNA complex some 200 bp downstream. Furthermore, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression. Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibility by pyrimidines of P1 promoter activity. The absence of both these effects in a double, deletion-duplication, mutant resulting in a net increase of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulatory site, but must be attributed to variations in the distance separating different protein binding sites.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Óperon , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Sequência de Bases , Proteínas de Escherichia coli , Metilação , Metiltransferases/genética , Dados de Sequência Molecular , Nucleotídeos de Pirimidina/genéticaRESUMO
The clinical features of exercise-induced anaphylaxis in 5 patients are described, with an accurate history, skin tests, IgE determinations and exercise tests. In these 5 patients we were able to distinguish three different forms of exercise-induced disease: exercise-induced anaphylaxis based upon an IgE-mediated allergy to foods, exercise-induced anaphylaxis without food allergy and cholinergic urticaria.
Assuntos
Anafilaxia/etiologia , Hipersensibilidade Alimentar/complicações , Esforço Físico , Adolescente , Adulto , Alérgenos , Feminino , Hipersensibilidade Alimentar/diagnóstico , Humanos , Masculino , Testes CutâneosRESUMO
Since 1983 the Netherlands Centre for Monitoring of Adverse Reactions to Drugs received 6 reports of anaphylaxis due to a chlorhexidine-containing lubricant used during cystoscopy or urinary catheterization. One case was described earlier. An accidental rechallenge in two patients was positive. In 5 cases, the causal relationship with chlorhexidine was confirmed by a positive reaction to intracutaneous testing. Although this adverse reaction is probably rare, medical practitioners should be aware of the possibility that a severe anaphylactic reaction may occur during use of these products.
Assuntos
Anafilaxia/induzido quimicamente , Clorexidina/efeitos adversos , Corticosteroides/administração & dosagem , Idoso , Anafilaxia/diagnóstico , Anafilaxia/tratamento farmacológico , Clemastina/administração & dosagem , Quimioterapia Combinada , Feminino , Humanos , Testes Intradérmicos , Masculino , Pessoa de Meia-Idade , Testes CutâneosAssuntos
Ornitina Carbamoiltransferase/isolamento & purificação , Ornitina Carbamoiltransferase/metabolismo , Pyrococcus furiosus/enzimologia , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Colorimetria/métodos , Estabilidade Enzimática , Temperatura Alta , Indicadores e Reagentes , Cinética , Ornitina Carbamoiltransferase/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , TermodinâmicaAssuntos
Cistite Intersticial/etiologia , Hipersensibilidade Alimentar/complicações , Hipersensibilidade Imediata/complicações , Hipersensibilidade Imediata/diagnóstico , Cistite Intersticial/diagnóstico , Diagnóstico Diferencial , Método Duplo-Cego , Hipersensibilidade Alimentar/diagnóstico , Humanos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
A group of genes regulated by arginine was found clustered in the order argF-ORF1-argC-argJ-ORF4 between other, as yet uncharacterized, open reading frames (ORFs). Transcription starts were identified immediately upstream from argF and ORF4. Arginine repressed transcription that was initiated at argF but induced transcription of ORF4. The functions of ORF1 and ORF4 are unknown, but analysis of the sequence of ORF4 suggests that it is a membrane protein, possibly involved in transport of arginine or a related metabolite. Mobility shift and DNase I footprinting have revealed specific binding of pure Escherichia coli ArgR to the promoter region of Thermus thermophilus argF. These results suggest that argF transcription is controlled by a repressor homologous to those characterized in enteric bacteria and bacilli. Thermus argF mRNA is devoid of Shine-Dalgarno (SD) sequences. However, downstream from the ATG start codon of argF and many other Thermus genes (with or without an SD box), sequences were found to be complementary to nucleotides 1392 to 1409 of Thermus 16S rRNA, suggesting that an mRNA-rRNA base pairing in this region is important for correct translation initiation.
Assuntos
Arginina/biossíntese , Proteínas de Bactérias , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Fases de Leitura Aberta , Thermus thermophilus/genética , Transcrição Gênica , Sequência de Aminoácidos , Arginina/genética , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Escherichia coli/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Thermus thermophilus/metabolismoRESUMO
A total of 360 patients with perennial allergic rhinitis were randomized in a placebo-controlled, dose-finding study comparing three concentrations (0.06%, 0.125%, and 0.25%) of a cetirizine nasal spray, administered three times a day for 2 weeks. The primary criterion of efficacy was the percentage of days with no or only mild symptoms of rhinitis (PDMax1), as evaluated by the patients. The median PDMax1 were 16.7%, 30.8%, 42.9%, and 26.7% for the placebo, 0.06%, 0.125%, and 0.25% groups, respectively. Although the global comparison among the four groups only approached statistical significance (P = 0.076), the difference (26.2%) between the placebo and 0.125% groups was clinically and statistically significant (P = 0.011). For the global evaluation by the investigator, the best results were seen in the 0.125% group (P = 0.03). The occurrence of adverse events did not differ among the four treatment groups and consisted mainly of nasal events, occurring in 22.5%, 17.1%, 12.9%, and 24.4% of the patients for the placebo, 0.06%, 0.125%, and 0.25% groups, respectively (P = 0.184). These results indicate that the 0.125% concentration is significantly better than placebo and offers the best therapeutic ratio.
Assuntos
Cetirizina/administração & dosagem , Rinite Alérgica Perene/tratamento farmacológico , Administração por Inalação , Adolescente , Adulto , Aerossóis , Idoso , Cetirizina/efeitos adversos , Cetirizina/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We have obtained mutants of Pyrococcus furiosus ornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15 degrees C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.
Assuntos
Adaptação Biológica/genética , Evolução Molecular , Mutação , Ornitina Carbamoiltransferase/genética , Pyrococcus furiosus/enzimologia , Estabilidade Enzimática , Teste de Complementação Genética , Cinética , Modelos Moleculares , Ornitina Carbamoiltransferase/metabolismo , Pyrococcus furiosus/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , TemperaturaRESUMO
Forty children underwent two different skin prick tests with allergen-coated Phazet needles and conventional skin prick tests using Pharmalgen extracts (100,000 BU/mL). Good correlation between both skin test methods was found with house dust mites (Rs = 0.61; P less than .001), timothy pollen (Rs = 0.77; P less than .001), and cat epithelium (Rs = 0.74, P less than .001). The correlation coefficient in the case of histamine was lower (Rs = 0.50; P less than .01) than that obtained from the allergens. Larger wheals were generated with Phazet than with conventional prick tests (median: 54.5 mm2 and 24.5 mm2, respectively; Wilcoxon signed rank test, P less than .0001). Comparison of wheal areas and specific IgE did not reveal large differences between the two skin test methods. In conclusion, Phazet, a recently introduced simple, standardized, skin prick test method can replace the conventional skin prick test for the most important allergens such as house dust mites, timothy pollen, and cat epithelium.
Assuntos
Alérgenos/imunologia , Histamina/imunologia , Testes Cutâneos/métodos , Adolescente , Criança , Epitopos , Feminino , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/imunologia , MasculinoRESUMO
We report the identification of Integration Host Factor (IHF) as a new element involved in modulation of P1, the upstream pyrimidine-specific promoter of the Escherichia coli K12 and Salmonella typhimurium carAB operons. Band-shift assays, performed with S-30 extracts of the wild type and a himA, hip double mutant or with purified IHF demonstrate that, in vitro, this factor binds to a region 300 bp upstream of the transcription initiation site of P1 in both organisms. This was confirmed by deletion analysis of the target site. DNase I, hydroxyl radical and dimethylsulphate footprinting experiments allowed us to allocate the IHF binding site to a 38 bp, highly A+T-rich stretch, centred around nucleotide -305 upstream of the transcription initiation site. Protein-DNA contacts are apparently spread over a large number of bases and are mainly located in the minor groove of the helix. Measurements of carbamoyl-phosphate synthetase (CPSase) and beta-galactosidase specific activities from car-lacZ fusion constructs of wild type or IHF target site mutants introduced into several genetic backgrounds affected in the himA gene or in the pyrimidine-mediated control of P1 (carP6 or pyrH+/-), or in both, indicate that, in vivo, IHF influences P1 activity as well as its control by pyrimidines. IHF stimulates P1 promoter activity in minimal medium, but increases the repressibility of this promoter by pyrimidines. These antagonistic effects result in a two- to threefold reduction in the repressibility of promoter P1 by pyrimidines in the absence of IHF binding. IHF thus appears to be required for maximal expression as well as for establishment of full repression. IHF could exert this function by modulating the binding of a pyrimidine-specific regulatory molecule.
Assuntos
Proteínas de Bactérias/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Proteínas de Ligação a DNA/metabolismo , Enterobacteriaceae/genética , Regiões Promotoras Genéticas/genética , Pirimidinas/metabolismo , Sequência de Bases , Sistema Livre de Células , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Mutagênese , Óperon , Salmonella typhimurium/genética , Deleção de SequênciaRESUMO
On a 4.7-kbp HindIII clone of Thermus strain ZO5 DNA, complementing an aspartate carbamoyltransferase mutation in Escherichia coli, we identified a cluster of four potential open reading frames corresponding to genes pyrR, and pyrB, an unidentified open reading frame named bbc, and gene pyrC. The transcription initiation site was mapped at about 115 nucleotides upstream of the pyrR translation start codon. The cognate Thermus pyr promoter also functions in heterologous expression of Thermus pyr genes in E. coli. In Thermus strain ZO5, pyrB and pyrC gene expression is repressed three- to fourfold by uracil and increased twofold by arginine. Based on the occurrence of several transcription signals in the Thermus pyr promoter region and strong amino acid sequence identities (about 60%) between Thermus PyrR and the PyrR attenuation proteins of two Bacillus sp., we propose a regulatory mechanism involving transcriptional attenuation to control pyr gene expression in Thermus. In contrast to pyr attenuation in Bacillus spp., however, control of the Thermus pyr gene cluster would not involve an antiterminator structure but would involve a translating ribosome for preventing formation of the terminator RNA hairpin. The deduced amino acid sequence of Thermus strain ZO5 aspartate carbamoyltransferase (ATCase; encoded by pyrB) exhibits the highest similarities (about 50% identical amino acids) with ATCases from Pseudomonas sp. For Thermus strain ZO5 dihydroorotase (DHOase; encoded by pyrC), the highest similarity scores (about 40% identity) were obtained with DHOases from B. caldolyticus and Bacillus subtilis. The enzyme properties of ATCase expressed from truncated versions of the Thermus pyr gene cluster in E. coli suggest that Thermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase complex.
Assuntos
Genes Bacterianos , Família Multigênica , Pirimidinas , Thermus/genética , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a lambda-ZAP genomic library. An ORF of 903 bp was found coding for a protein of Mr 33,200. The coding region has a very high overall G+C content of 68.0%. T. thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus furiosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on delta-N-phosphonoacetyl-L-ornithine-Sepharose. The native enzyme has an Mr of about 110,000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. T. thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a Km(app) of 0.10 mM for both substrates. The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around Km values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65 degrees C and the activation energy 39.1 kJ/mol. The enzyme is highly thermostable. In the presence of its substrates the half-life time was several hours at 85 degrees C. Ionic and hydrophobic interactions contribute to the stability. The expression of T. thermophilus OTC was negatively regulated by arginine.