RESUMO
Metal-transporting P-type ATPases were recently proposed to constitute a newly emerged sub-family of cation-transporting P-type ATPases, and are known to occur widely in prokaryotes and eukaryotes. However, no instance has been reported for higher plants. A cDNA clone encoding a metal-transporting P-type ATPase was thus searched for, if present, and was identified in Arabidopsis thaliana. The amino acid sequence, predicted from the determined nucleotide sequence for the cloned cDNA, shows all the critical features common to known metal-transporting P-type ATPases. This plant P-type ATPase has a typical metal-binding motif at its N-terminal portion. The newly isolated Arabidopsis gene, named PAA1, provides us with the first instance of putative metal-transporting P-type ATPases in higher plants. Some results of genomic analyses for this gene are also presented.
Assuntos
Adenosina Trifosfatases/genética , Arabidopsis/enzimologia , DNA Complementar/genética , Genes de Plantas , Sequência de Aminoácidos , Arabidopsis/genética , Sítios de Ligação , Clonagem Molecular , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
The Escherichia coli nucleoid protein, H-NS, functions as a global regulator for expression of a wide variety of genes. We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-binding, respectively. However, identification of the presumed dimerization domain of H-NS and its functional significance was elusive. To address this particular issue, we first examined a set of N-terminally or C-terminally truncated forms of H-NS, in terms of their so-called dominant-negative effect on the in vivo function of the wild-type H-NS. The results showed that certain truncated forms exhibit such a dominant-negative effect, but others did not. As judged by the results of the dominant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization. This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS. Furthermore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, which was expected to be specifically defective in dimerization. This mutant had an amino acid substitution at position 30 (Leu30 to Pro) in N91 consisting of the N-terminal 91 amino acids of H-NS. This mutant was indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS. When this particular single amino acid substitution was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a transcriptional repressor. These findings collectively provided us with evidence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/química , Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/química , Sequência de Aminoácidos , Western Blotting , Reagentes de Ligações Cruzadas/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Dimetil Suberimidato/metabolismo , Escherichia coli/genética , Óperon Lac/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Deleção de Sequência , Transcrição Gênica , Transfecção , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively. We first isolated a number of hns mutations which resulted in derepression of the proVWX operon. These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS. The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS. This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties. Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA. The N-terminal domain was suggested to be involved in the ability to repress transcription. Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity. None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization. These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , DNA Bacteriano/genética , Immunoblotting , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
A polypeptide with an apparent molecular mass of 23 kDa was identified, that exhibited an affinity to a 491-bp DNA derived from one of the Schizosaccharomyces pombe centromeric DNAs (cen1). After determining its N-terminal amino acid (aa) sequence, a Sz. pombe genomic DNA encompassing the coding sequence of the isolated protein was cloned, and a 2.3-kb genomic DNA region sequenced. Further sequence analysis of cDNA clones, originating from this particular genomic region, confirmed the existence of an open reading frame with a short intron, which encodes a 409-aa protein with striking homology to eukaryotic elongation factor-1 gamma.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Fatores de Alongamento de Peptídeos/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Fator 1 de Elongação de Peptídeos , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Previously, Arabidopsis thaliana was shown to possess a set of response regulators (ARR-series), which are implicated in the prokaryotic type of signal transduction mechanism, generally referred to as the His-Asp phosphorylay. Among them, ARR4 is a typical phospho-accepting response regulator, whose expression was recently demonstrated to be rapidly induced by a cytokinin-treatment of the plant. To gain insight into the presumed His-Asp phosphotransfer signaling mechanism as well as the role of ARR4 in this higher plant, in this study we adopt the widely used yeast two-hybrid system, and report the identification of an Arabidopsis protein that has an ability to interact physically with the cytokinin-inducible ARR4 response regulator.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histidina/metabolismo , Células Híbridas , Dados de Sequência Molecular , Fosforilação , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Leveduras/genéticaRESUMO
We examined cytokinin and nitrate responsiveness in gene expression of five distinct response regulator homologs (ARR3-ARR7) in the leaves of nitrogen-starved Arabidopsis plants. The transcripts accumulated after spraying the shoots with t-zeatin. The induction of accumulation was highly specific for cytokinins. The transcripts also accumulated by supply of nitrate to the culture medium. These findings suggest that ARRs are involved in inorganic nitrogen signal transduction mediated by cytokinin as in the case of ZmCip1, a response regulator homolog recently identified in maize.
Assuntos
Arabidopsis/efeitos dos fármacos , Citocininas/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Nitratos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/biossíntese , Folhas de Planta/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA de Plantas/biossínteseRESUMO
The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
The three-dimensional structure of the C-terminal domain (47 residues) obtained from the hydrolysis of H-NS protein with bovine trypsin was determined by NMR measurements and distance geometry calculations. It is composed of an antiparallel beta-sheet, an alpha-helix and a 3(10)-helix which form a hydrophobic core, stabilizing the whole structure. This domain has been found to bind to DNA. Possible DNA binding sites are discussed on the basis of the solution structure of the C-terminal domain of H-NS.
Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Sequência de Bases , Escherichia coli/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Fragmentos de Peptídeos , Estrutura Terciária de Proteína , SoluçõesRESUMO
To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.
Assuntos
Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Leveduras/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Proteínas Fúngicas/genética , Regulação Bacteriana da Expressão Gênica , Genes Supressores , Dados de Sequência Molecular , Mutação , Plasmídeos , Mapeamento por RestriçãoRESUMO
The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. The dnaJ- cbpA- double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ- cbpA- mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD, the gene that encodes the major sigma 70. The biological implications of this finding are examined and discussed.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Chaperonas Moleculares/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Amplificação de Genes , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Mutação , Fator sigma/genética , Supressão GenéticaAssuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Transcrição Gênica , Proteínas da Membrana Bacteriana Externa/biossíntese , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , DNA Super-Helicoidal/isolamento & purificação , Proteínas de Ligação a DNA/biossíntese , Eletroforese em Gel de Poliacrilamida/métodos , Radioisótopos de Fósforo , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Moldes Genéticos , Uridina Trifosfato/metabolismoRESUMO
Protein translocation across the Escherichia coli cytoplasmic membrane may consist of several temporally or topographically distinct steps. Although early events in the translocation pathway have been characterized to some extent, the mechanisms responsible for the trans-bilayer movement of a polypeptide are only poorly understood. This article reports on our attempts to dissect the translocation pathway in vivo. A processed form of maltose-binding protein (MBP) was detected in the spheroplasts of secY and secA temperature-sensitive mutant cells that had been pulse-labeled at the permissive temperature (30 degrees C). This species of molecule was found to have an electrophoretic mobility identical to that of the mature MBP, but a considerable fraction of it was inaccessible to externally added protease. It had not attained the protease-resistant conformation characteristically observed for the exported mature protein. The radioactivity associated with this species decreased during chase and was presumably converted into the exported mature form, a process that required energy, probably the proton motive force, as demonstrated by its inhibition by an energy uncoupler. The spheroplast-associated processed form was more predominantly observed in the presence of a low concentration of chloramphenicol. A similar intermediate was also detected for beta-lactamase in wild-type cells. These results suggest that in a late phase of translocation, the bulk of the polypeptide chain can move through the membrane in the absence of the covalently attached leader peptide, and the secA-secY gene products are somehow involved in this process. We termed the processed intermediates processed immature forms.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Transporte de Monossacarídeos , Mutação , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Cloranfenicol/farmacologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Cinética , Maltose/metabolismo , Proteínas Ligantes de Maltose , Proteínas de Membrana/genética , Fragmentos de Peptídeos/isolamento & purificação , TripsinaRESUMO
Escherichia coli genes were cloned onto a multicopy plasmid and selected by the ability to restore growth and protein export defects caused by a temperature-sensitive secY or secA mutation. When secA51 was used as the primary mutation, only clones carrying groE, which specifies the chaperonin class of heat shock protein, were obtained. Selection using secY24 yielded three major classes of genes. The first class encodes another heat shock protein, HtpG; the most frequently obtained second class encodes a neutral histonelike protein, H-NS; and the third class, msyB, encodes a 124-residue protein of which 38 residues are acidic amino acids. Possible mechanisms of suppression as well as the significance and limitations of the multicopy suppression approach are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Teste de Complementação Genética , Proteínas de Choque Térmico HSP90 , Proteínas de Choque Térmico/genética , Supressão Genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico Ativo/genética , Chaperoninas , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , Família Multigênica , Plasmídeos/genética , Processamento de Proteína Pós-TraducionalRESUMO
The H-NS protein is a major constituent of the Escherichia coli nucleoid structure and is implicated in the compact organization of the chromosome. Based on recent genetic evidence, this protein appears to influence the transcription of a variety of apparently unlinked genes on the chromosome, although the underlying molecular mechanism is not fully understood. In this study, we carried out a series of in vitro transcription assays including purified H-NS with special reference to the osmotically inducible proV promoter of the proVWX operon (or proU), whose expression is known to be derepressed by lesions of the hns (osmZ) gene. Here, H-NS was revealed to selectively inhibit an early step(s) of proV transcription initiation through its direct binding to the promoter region. It was thus demonstrated that H-NS functions directly as a transcriptional repressor. Under the in vitro conditions used, this in vitro inhibitory effect of H-NS was affected by changes in the superhelical density of template DNAs and more significantly by the concentration of potassium (K+) ions. These results are also discussed with regard to the mechanism underlying regulation of the proV promoter in response to the medium osmolarity.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , DNA Super-Helicoidal/metabolismo , Escherichia coli/metabolismo , Osmose , Potássio/metabolismo , Regiões Promotoras Genéticas , Moldes GenéticosRESUMO
The mechanism by which the Escherichia coli proV promoter is activated more than 100-fold in response to the medium osmolarity, without the help of any known trans-acting activators, is not yet fully understood. In this context, it has recently begun to be realized that structural features, not the primary sequences, of cis-acting DNA elements may be important for transcriptional regulation in prokaryotes. From this point of view, in this study the proV promoter was characterized by constructing a series of spacer-insertion mutants in a proV-lacZ fusion on the chromosome. Here it was found that the upstream cis-acting sequence must be positioned stereospecifically with respect to the principal -35 and -10 regions for the proV promoter to be fully activated. In this regard, it was suggested that an overall DNA structure, particularly DNA curvature, is an important cis-acting parameter for activation of the proV promoter.
Assuntos
DNA Bacteriano/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Membrana Transportadoras , Proteínas Periplásmicas de Ligação , Regiões Promotoras Genéticas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Insercional , Conformação de Ácido Nucleico , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The sigma(S) (or sigma(38)) subunit of RNA polymerase, encoded by the rpoS gene, is a crucial regulator in the transcriptional control of a set of genes under stressful conditions, such as nutrient starvation. The expression of rpoS is regulated in a complex manner at the levels of transcription, translation, and stability of the product. Although a number of factors involved in the regulation of rpoS expression have been identified, the underlying molecular mechanisms are not fully understood. In this study, we identified the Crr (or EIIA(Glc)) protein as a novel factor that plays an important role not only in the transcriptional control but also in the translational control of rpoS expression. Crr is an important component in glucose uptake through the well-characterized phosphoenolpyruvate:carbohydrate phosphotransferase system. The results of a series of genetic analyses revealed that Crr negatively controls rpoS translation and transcription. The observed transcriptional control by Crr appears to be mediated by cyclic AMP. However, it was found that Crr negatively controls rpoS translation rather directly. These results suggest a possible linkage between the control of rpoS expression and carbon metabolism.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fator sigma/genética , Proteínas de Transporte/genética , AMP Cíclico/metabolismo , Epistasia Genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Fatores Hospedeiros de Integração , Mutagênese Insercional , Biossíntese de Proteínas , Transcrição GênicaRESUMO
H-NS is a major constituent of the Escherichia coli nucleoid. Expression of the major outer membrane proteins, OmpC and OmpF, is influenced by hns mutations such that OmpC expression increases whereas OmpF expression decreases irrespective of the osmolarity of the medium (K. A. Graeme-Cook, G. May, E. Bremer, and C. F. Higgins, Mol. Microbiol. 3:1287-1294, 1989). In this study we show that the effect of an hns::neo mutation on OmpF expression is largely diminished in a deletion mutant carrying the micF gene that encodes the ompF mRNA-specific antisense RNA. In addition, the micF transcript levels in the hns::neo mutation are high compared with transcript levels in wild-type cells. On the basis of these results, we provide evidence for a link between OmpC/OmpF expression and the regulatory function of H-NS. We suggest that H-NS most likely affects OmpC expression directly at the level of transcription, but OmpF expression is indirectly regulated by micF antisense RNA.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Antissenso , RNA Bacteriano/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
In Escherichia coli, recent intensive studies revealed that expression of a certain subset of genes is under the control of the stationary phase-specific sigma factor, sigma S, which is encoded by the rpoS gene. Since sigma S functions predominantly under certain growth conditions, its activity and/or cellular content has accordingly to be tightly controlled, however, the underlying molecular mechanism is at present unclear. We previously demonstrated that expression of the cbpA gene, encoding an analogue of the DnaJ molecular chaperone, is largely dependent on sigma S function. Here we have found that a mutational lesion of the hns gene, which encodes one of the well-characterized nucleoid proteins, H-NS, affects the cellular content of sigma S remarkably and consequently affects the expression of cbpA. Enhanced accumulation of sigma S in hns deletion cells was particularly observed in the logarithmic growth phase and was demonstrated to result from an elevated translational efficiency of rpoS mRNA and also from an increased stability of newly synthesized sigma S. Although H-NS is known to influence the transcription of a number of apparently unlinked genes on the chromosome, in this study we provide a novel instance in which H-NS is deeply implicated in post-transcriptional regulation(s) of the expression of rpoS. As to physiological relevance, it was also demonstrated that hns deletion cells exhibit an extreme thermotolerance even in the logarithmic growth phase, presumably because of the enhanced accumulation of sigma S.