RESUMEN

Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves beta-1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 A shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa-N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical "chair" to "boat" conformational change on the sugar residue bound to the -1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of -1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed "substrate assisted" catalysis.

Asunto(s)

Quitinasas/química , Quitinasas/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Serratia marcescens/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Conformación de Carbohidratos , Secuencia de Carbohidratos , Catálisis , Clonación Molecular , Cristalografía por Rayos X/métodos , Escherichia coli , Isopropil Tiogalactósido/química , Isopropil Tiogalactósido/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Sensibilidad y Especificidad , Programas Informáticos , Especificidad por Sustrato

RESUMEN

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of lambda DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

Asunto(s)

Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , ADN/química , Proteínas Bacterianas/genética , Bacteriófago lambda/genética , ADN Viral/química , Proteínas de Unión al ADN/genética , Elasticidad , Factores de Integración del Huésped , Mutagénesis Sitio-Dirigida

RESUMEN

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (sigma(32)) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH(1) is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36-122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Factor sigma , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Bradyrhizobium/enzimología , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , beta-Galactosidasa/metabolismo

RESUMEN

The Escherichia coli heat shock sigma factor sigma32 (RpoH) is rapidly degraded under non-stress conditions. The integrity of the DnaK chaperone machinery and the ATP-dependent FtsH protease are required for sigma32 proteolysis in vivo. Bradyrhizobium japonicum expresses three sigma32-type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coli sigma32 stability. In E. coli C600 (wild-type), the half-lives of sigma32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild-type. Proteolysis of RpoH3 was FtsH-independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of sigma32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH-mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.

Asunto(s)

Bradyrhizobium/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Choque Térmico/metabolismo , Factor sigma , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Proteínas Bacterianas/metabolismo , Bradyrhizobium/metabolismo , Cloranfenicol/farmacología , Escherichia coli/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Calor , Immunoblotting , Isopropil Tiogalactósido/farmacología , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Péptidos/inmunología , Péptidos/metabolismo , Mutación Puntual , Proteínas Recombinantes/metabolismo , Temperatura , Factores de Tiempo , Factores de Transcripción/genética

RESUMEN

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase -substrate co-crystals. We found that both residues are essential for chitobiase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues.

Asunto(s)

Acetilglucosamina/metabolismo , Acetilglucosaminidasa/química , Acetilglucosaminidasa/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Mutación/genética , Serratia marcescens/enzimología , Acetilglucosamina/análogos & derivados , Acetilglucosaminidasa/genética , Sustitución de Aminoácidos , Ácido Aspártico/genética , Sitios de Unión , Catálisis , Secuencia Conservada/genética , Cristalización , Cristalografía por Rayos X , Ácido Glutámico/genética , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Serratia marcescens/genética , Relación Estructura-Actividad , Termodinámica

RESUMEN

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).

Asunto(s)

Proteínas Bacterianas/química , Escherichia coli/enzimología , Proteínas de la Membrana/química , Factor sigma , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Secuencia Conservada/genética , Reactivos de Enlaces Cruzados/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Factores de Transcripción/metabolismo , Tripsina/metabolismo , Proteínas Virales

RESUMEN

FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known. The enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the lambda CIII gene product. In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage lambda CII protein. It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site. beta-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis. The majority of the peptides produced were 13 to 20 residues long.

Asunto(s)

Proteínas Bacterianas/metabolismo , Bacteriófago lambda , Endopeptidasas/metabolismo , Escherichia coli/enzimología , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Caseínas/metabolismo , Proteínas de Escherichia coli , Datos de Secuencia Molecular , Especificidad por Sustrato , Factores de Transcripción/aislamiento & purificación , Proteínas Virales

RESUMEN

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

Asunto(s)

Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Tilacoides/enzimología , Proteasas ATP-Dependientes , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Proteínas de Arabidopsis , Caseínas/metabolismo , Dominio Catalítico , Cationes/farmacología , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Luz , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Metaloendopeptidasas/genética , Metaloendopeptidasas/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Complejo de Proteína del Fotosistema II , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Tilacoides/efectos de los fármacos

RESUMEN

An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype.

Asunto(s)

Proteínas Bacterianas/genética , Colicinas/farmacología , Escherichia coli/genética , Proteínas de la Membrana/genética , Mutación , Proteasas ATP-Dependientes , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/fisiología , Clonación Molecular , Escherichia coli/efectos de los fármacos , Escherichia coli/virología , Proteínas de Escherichia coli , Regulación Viral de la Expresión Génica , Lisogenia/fisiología , Proteínas de la Membrana/metabolismo , Fenotipo , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Transducción Genética , Proteínas Virales

RESUMEN

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.

Asunto(s)

Proteínas de la Cápside , Cápside/biosíntesis , Cápside/aislamiento & purificación , Chaperonina 10/genética , Chaperonina 60/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Poliomavirus/genética , Cápside/genética , Chaperonina 10/biosíntesis , Chaperonina 10/metabolismo , Chaperonina 60/biosíntesis , Chaperonina 60/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Líquido Intracelular/metabolismo , Virión/genética , Virión/metabolismo

RESUMEN

We have previously identified a UP element in the phage lambda PL promoter, centred at position -90 from the transcription start site. Integration host factor (IHF), a heterodimeric DNA-binding and -bending protein, binds upstream of the lambda PL promoter in a region overlapping the UP element. Stimulation of transcription by IHF requires an intact alphaCTD and affects the initial binding of RNA polymerase to the promoter. We propose a model for the stimulation of PL by IHF in which IHF bends the DNA to bring the distal UP sequence in closer proximity to the promoter core sequences to allow the docking of the alphaCTD of RNA polymerase. Furthermore, IHF may also participate in protein-protein interactions with the alphaCTD. In support of this model, we found that alanine substitutions in alphaCTD at positions 265, 268, 270 and 275 reduced PL promoter activity. Mutations in the IHF DNA binding site, as well as IHF mutant proteins exhibiting a decreased ability to bend the DNA, were both defective in stimulating the PL promoter. In addition, some of the mutated IHF residues are clustered at a protein surface that interacts with the UP DNA sequence. These residues may also participate in protein-protein interactions with the alphaCTD.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , ADN/química , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Factores de Integración del Huésped , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Conformación Proteica , Transcripción Genética

RESUMEN

Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

Asunto(s)

Proteínas de la Cápside , Cápside/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Regiones Promotoras Genéticas , Virus 40 de los Simios/fisiología , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Células HeLa , Humanos , Nucleosomas/fisiología , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos , Virus 40 de los Simios/genética

RESUMEN

A shift in growth temperature from 37 degrees C to 15 degrees C leads to a dramatic increase in the level of CspA, the major cold shock protein of Escherichia coli. To investigate the molecular basis of this induction, we considered the relevance of transcriptional and posttranscriptional controls by analyzing the steady-state levels of transcripts and the expression of reporter genes in cells carrying a set of cspA promoter fragments of variable length fused to lacZ or cat genes. We demonstrate that: (i) the core cspA promoter (from -40 to +16) responds to cold shock and a mutation at -36 increases the relative activity of the promoter at low temperature by threefold; (ii) the sequences upstream of -40 have a positive effect on expression at 37 degrees C, but no effect on the cold shock response; (iii) by virtue of their influence on mRNA stability, the downstream sequences (from +81 to +165) reduce expression at 37 degrees C and increase the intensity of the cold shock response; (iv) mutations in the GCACATCA and CCAAT motifs, present at +1/-4 and between the -10 and -35 elements, respectively, do not affect the cold shock response of the cspA promoter; (v) following cold shock, a modification of the protein synthetic machinery takes place that allows preferential translation of cspA mRNA relative to the non-cold shock cat and lacZ mRNAs. The quantitatively modest transcriptional activation shown by the core promoter of cspA following cold shock suggests that transcriptional activation can significantly contribute to cold shock induction only when coupled to posttranscriptional controls, such as alterations in mRNA stability and the translational apparatus.

Asunto(s)

Proteínas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Adaptación Fisiológica , Frío , Mutagénesis , Procesamiento Proteico-Postraduccional

Asunto(s)

ADN Bacteriano/metabolismo , Escherichia coli/genética , Secuencias Hélice-Giro-Hélice , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Células Procariotas , Conformación Proteica , Proteínas Represoras/química , Transactivadores/química

RESUMEN

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

Asunto(s)

Bacteriófago lambda/fisiología , Factores de Transcripción/fisiología , Bacteriófago lambda/genética , Frío , Replicación del ADN , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Lisogenia , Mutación , Regiones Promotoras Genéticas , Temperatura , Factores de Transcripción/genética , Transcripción Genética , Proteínas Virales/fisiología , Replicación Viral

RESUMEN

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage lambda CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage lambda. Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor sigma32. Proteolysis of sigma32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of sigma32. Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6-7 nm.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago lambda , Escherichia coli/enzimología , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/ultraestructura , Proteínas Bacterianas/ultraestructura , Endopeptidasas/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli , Proteínas de la Membrana/ultraestructura , Proteínas Virales

RESUMEN

SV40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. In the present study, the three SV40 capsid proteins, VP1, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells. Their co-production led to spontaneous assembly of SV40-like particles. Nuclear extracts containing the three proteins were allowed to interact with purified SV40 DNA, or with plasmid DNA produced and purified from Escherichia coli. The experiments demonstrated a physical association between the DNA and capsid proteins, protection from DNase I digestion, and the formation of infectious particles. The results indicate that intact, supercoiled DNA is being packaged and transmitted into the target cells. The transmitted DNA is biologically functional in gene expression and replication. The process, which utilizes naked DNA, is not dependent on the SV40 packaging signal ses. The procedure allows packaging of plasmids significantly larger than SV40 and permits the inclusion of potent regulatory signals, such as beta-globin locus control region (LCR) elements. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use.

Asunto(s)

Cápside/genética , Vectores Genéticos , Virus 40 de los Simios/fisiología , Animales , Proteínas de la Cápside , Extractos Celulares , Línea Celular , Núcleo Celular , Chlorocebus aethiops , ADN Viral , Genes Virales , Terapia Genética , Humanos , Plásmidos , Proteínas Recombinantes de Fusión/genética , Virus 40 de los Simios/genética , Spodoptera/citología , Virión , Ensamble de Virus

RESUMEN

The C-terminus of the alpha subunit of Escherichia coli RNA polymerase is known to function in transcriptional activation at certain promoters. This region was previously shown to be necessary for full activation of the pE promoter by the phage lambda CII protein in vitro. In this work we investigated the inability of phage lambda to follow the lysogenic pathway in cells carrying the point mutation rpoA341 (a change of lysine 271 to glutamic acid). We found that neither overexpression of the cII gene nor stabilisation of the CII protein by the can1 mutation or by cIII gene overexpression was able to suppress the block in lysogenisation. In contrast, the lambda cin1 phage, which carries a CII-independent promoter for the expression of the cI gene, was able to efficiently lysogenise the rpoA341 mutant strain. Furthermore, the rpoA341 mutation prevented the activation of pE-lacZ and pI-lacZ transcriptional fusions by CII. Therefore we conclude that transcriptional activation by the cII gene product is abolished by the rpoA341 mutation, most probably due to impaired interaction between the CII activator and mutant RNA polymerase. The inability of RNA polymerase to respond to CII results in the impairment of lysogenisation of the rpoA341 mutant by phage lambda.

Asunto(s)

Bacteriófago lambda/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/genética , Mutación , Transcripción Genética , Proteínas Virales

RESUMEN

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions.

Asunto(s)

Quitinasas/genética , Enterobacter/enzimología , Secuencia de Aminoácidos , Antifúngicos/farmacología , Secuencia de Bases , Quitinasas/química , Quitinasas/farmacología , Clonación Molecular , Enterobacter/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Rhizoctonia/crecimiento & desarrollo

RESUMEN

Using a bacteriophage lambda complementation system in Escherichia coli, we cloned genes encoding subunits of the heterodimeric DNA binding/bending protein, integration host factor, from the bovine pathogen, Pasteurella haemolytica. Complementation of ihfA and ihfB mutations in E. coli demonstrated that the P. haemolytica gene products form functional heterologous heterodimers. The ihfA and ihfB genes encode polypeptides predicted to be 99 and 93 amino acids long, respectively, and are very similar to integration host factor subunits from other Gram-negative bacteria, although phylogenetic analysis indicated that the P. haemolytica sequences are distantly related to those from other bacteria. Most significant amino acid differences were restricted to the amino-terminal domains of the predicted peptides.

Asunto(s)

Proteínas Bacterianas/genética , Mannheimia haemolytica/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Biblioteca de Genes , Genes Bacterianos/genética , Prueba de Complementación Genética , Factores de Integración del Huésped , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido