RESUMO
Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not. The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/citologia , Fosfolipídeos/deficiência , Supressão Genética/genética , Substituição de Aminoácidos/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Divisão Celular , Cefalexina/farmacologia , Cromossomos Bacterianos/genética , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Citometria de Fluxo , Concentração de Íons de Hidrogênio , Complexo de Reconhecimento de Origem , Fosfolipídeos/química , Plasmídeos/genética , Estrutura Terciária de Proteína , Rifampina/farmacologia , Deleção de Sequência/genética , Transformação Bacteriana , Proteínas Virais/genéticaRESUMO
CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.
Assuntos
Proteínas de Bactérias , Replicação do DNA/genética , Proteínas de Escherichia coli , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular/genética , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Complexo de Reconhecimento de Origem , RNA Bacteriano/metabolismo , Proteínas Virais/genéticaRESUMO
DNA replication in Escherichia coli is controlled at the initiation stage, possibly by regulation of the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication. Accumulating evidence, both in vitro and in vivo, that supports the importance of membrane phospholipids influencing the initiation activity of DnaA is reviewed.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Concentração de Íons de Hidrogênio , Estrutura Terciária de Proteína , Origem de Replicação/genética , RepliconRESUMO
Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Origem de Replicação , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Especificidade de Anticorpos , Proteínas de Bactérias/imunologia , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromossomos Bacterianos , Crioultramicrotomia , Proteínas de Ligação a DNA/imunologia , Escherichia coli/genética , Escherichia coli/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Microscopia Imunoeletrônica/métodos , CoelhosRESUMO
DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Histidina , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia de Afinidade , Replicação do DNA , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Bacterianos , Peptídeos/química , Peptídeos/genética , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/fisiologia , Fosfolipídeos/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Nucleotídeos/metabolismo , Concentração Osmolar , Espectrometria de Fluorescência , Eletricidade Estática , UltracentrifugaçãoRESUMO
The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA. DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication. The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein. DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication. In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner. Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.
Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/metabolismo , Sistema Livre de Células/metabolismo , Escherichia coli , Modelos Genéticos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Origem de Replicação , Análise de SequênciaRESUMO
DnaA protein, the initiator protein of E. coli chromosomal replication, can be rejuvenated from an inactive ADP form to active ATP-DnaA protein by acidic phospholipids in a fluid bilayer. Cross-linking studies with the photoactivable phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin -3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine reveal insertion of DnaA protein into the hydrophobic region of the bilayer; this insertion is accompanied by membrane-mediated dissociation of the tightly bound allosteric nucleotides ADP and ATP. Photolabeling of DnaA protein occurred with membrane properties that resembled those needed for reactivation of ADP-DnaA protein; efficient labeling of DnaA protein was observed only when the lipid analog was incorporated into anionic vesicles and the temperature during treatment was above the gel to liquid crystalline phase transition. Predominant hydrophobic photolabeling was localized within a single region of DnaA protein, a region that contains putative amphipathic helices and has been shown to contain information essential for functional interaction with membranes.
Assuntos
Nucleotídeos de Adenina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Reagentes de Ligações Cruzadas , Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli , Fluidez de Membrana , Mapeamento de Peptídeos , Marcadores de Fotoafinidade , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.
Assuntos
Proteínas de Transporte/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Origem de Replicação , Proteínas de Bactérias/metabolismo , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação , Fatores Hospedeiros de IntegraçãoRESUMO
The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaA protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.
Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Quimotripsina/metabolismo , Gangliosídeos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Fosfolipídeos , Origem de ReplicaçãoRESUMO
The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaK protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.
Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Quimotripsina/metabolismo , Gangliosídeos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Fosfolipídeos/metabolismo , Origem de Replicação , Tripsina/metabolismoRESUMO
Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30 degrees C. Whereas purified wild-type DnA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild-type protein. In a system reconstituted with purified proteins at 30 degrees C, the mutant protein initiated replication of single-stranded DNA that contains a DnA-binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. Thermal stabilities of wild-type DnA and DnaAcos activities were comparable. Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Cromossomos Bacterianos , Ligação Proteica , Origem de Replicação/genéticaRESUMO
DnaA protein loses the capacity to initiate chromosomal replication when treated with a soluble cell extract. This inactivation depends upon DNA and hydrolyzable ribonucleoside triphosphate. The extract does not affect the activities of other replicative proteins or the ability of DnaA to initiate replication of single-stranded DNA that contains a DnaA-binding hairpin, indicating that the inhibitory effect is specific for the action of DnaA at oriC. Gel filtration experiments implicate a 150-kDa factor as being responsible. Mutant DnaAcos protein, which causes overinitiation in vivo, is insensitive to the inactivating factor, suggesting a requirement for this negative control in vivo. We propose that a soluble factor controls initiation through down-regulation of DnaA protein.
Assuntos
Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos , Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/genética , Trifosfato de Adenosina/farmacologia , Cromatografia DEAE-Celulose , Cromatografia em GelAssuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Origem de Replicação , Sistema Livre de Células , Cromossomos Bacterianos , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Cinética , Radioisótopos de Fósforo , Plasmídeos , Técnica de Diluição de Radioisótopos , Ribonuclease H/metabolismo , Moldes Genéticos , Nucleotídeos de Timina/metabolismoRESUMO
Regulation of the eukaryotic cell cycle involves calcium- and lipid-stimulated kinases acting on cytoskeletal structures; there are two principal reasons for supposing that the regulation of the prokaryotic cell cycle may be fundamentally the same. First, evidence for their fundamental difference is still missing and, second, evidence for prokaryotic homologues of eukaryotic cell cycle proteins is accumulating. Such proteins include those involved in calcium regulation, such as calmodulin and calcium-dependent kinases, and those involved in lipid regulation, such as protein kinase C. Proteins identified as candidates for cytoskeletal elements now include MukB, a putative contractile protein responsible for chromosome segregation, and FtsZ, the key constituent of the "cytokinetic" ring. These similarities allow the application of powerful prokaryotic model systems to one of biology's most profound, complex and urgent problems: the nature of the regulation of the eukaryotic cell cycle.
Assuntos
Ciclo Celular/fisiologia , Células Eucarióticas/citologia , Células Procarióticas/fisiologia , Animais , Proteínas do Citoesqueleto/metabolismo , Modelos Biológicos , Proteínas Quinases/metabolismoRESUMO
The ppk gene encoding polyphosphate kinase (PPK), the enzyme in Escherichia coli that makes long chains of polyphosphate (polyP) reversibly from ATP, was disrupted by insertion of a kanamycin resistance gene. Expression of the exopolyphosphatase gene (ppx) immediately downstream of ppk in the operon was likewise disrupted. Cells were also transformed with a high-copy-number plasmid bearing ppk. Genetically altered polyP levels were estimated in cell extracts by the PPK conversion of ADP to ATP. PolyP levels (microgram/10(11) cells) near 2.0 were reduced in the ppk(-)-ppx- mutants to 0.16 and increased more than 100-fold (e.g. 220) in cells transformed with multiple copies of ppk. Mutant cells, lacking the long polyP chains, showed a growth lag following dilution of a stationary-phase culture. PolyP-deficient cells exhibit a striking phenotype in their failure to survive in stationary phase and loss of resistance to heat (55 degrees C) and to oxidants (42 mM H2O2). High polyP levels are also associated with reduced survival.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Trifosfato de Adenosina/metabolismo , Escherichia coli/crescimento & desenvolvimento , Genes Bacterianos , Cinética , Plasmídeos , Mapeamento por Restrição , Especificidade da EspécieRESUMO
Acidic phospholipids in a fluid phase dissociate ADP or ATP tightly bound to DnaA protein and, in the presence of ATP and DNA, can restore an inactive ADP form to full activity (Sekimizu, K., and Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135). Further studies of the interactions between DnaA protein and lipids have used two functional assays: 1) release of ADP or ATP from DnaA and 2) DNA replication upon rejuvenation of an inactive ADP-DnaA protein complex. Among a variety of phospholipids tested were pure synthetic compounds and the mixtures from Escherichia coli auxotrophs (fabA), which are unable to synthesize unsaturated fatty acids and can be supplemented with different acyl derivatives. Fatty acid composition was determined by gas-liquid chromatography and membrane fluidity by fluorescence spectroscopy using 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid requirements of DnaA protein were shown to be: 1) phospholipids in a fluid phase (i.e. above the transition temperature), 2) a charged polar head group, 3) a lamellar phase (i.e. hexagonal II structures were inactive), and 4) a certain degree of fluidity imparted by the fatty acids esterified to the glycerol backbone. This conclusion was based on the incorporation of: 1) cholesterol, known to increase the packing of lipids, or 2) a branched fatty acyl derivative, which exhibits a fluidizing effect similar to that of a cis double bond. Both agents demonstrated that membrane fluidity is required for DnaA protein function in vitro, consistent with early studies of chromosome initiation in growing cells.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Fluidez de Membrana , Fosfolipídeos/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Escherichia coli/genética , Esterificação , Ácidos Graxos Insaturados/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas LipídicasRESUMO
DnaA protein and the Escherichia coli chromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication. We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex. One distinctive structure could be correlated with activity and localized to oriC, whilst several others could not. Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex. Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex. Mutagenesis of several DNA sequence elements in oriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex. As judged by electron microscopy and by functional assays, the region containing the four 9-mer dnaA boxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening of oriC, were not.
Assuntos
Proteínas de Bactérias/ultraestrutura , Cromossomos Bacterianos/ultraestrutura , Replicação do DNA , DNA Bacteriano/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Escherichia coli/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Análise Mutacional de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Recombinante/metabolismo , DNA Recombinante/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Conformação ProteicaRESUMO
A gene, ppx, that encodes a novel exopolyphosphatase of 513 amino acids (58,133 Da) was found downstream of the gene for polyphosphate kinase, ppk. Transcription of the ppx gene depends on the ppk promoters, indicating a polyphosphate (polyP) operon of ppk and ppx. Exopolyphosphatase, purified to homogeneity from overproducing cells, is judged to be a dimer of 58-kDa subunits. Orthophosphate is released processively from the ends of polyP approximately 500 residues long, but chains of approximately 15 residues compete poorly with polyP as substrate; ATP is not a substrate. Mg2+ (1 mM) and a high concentration of K+ (175 mM) support optimal activity.