RESUMEN
Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s(20,w)(0) were determined to be 3.64 and 5.51±0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5±0.2 Å and a maximum dimension of ~130 Å. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.
Asunto(s)
Proteínas Arqueales/química , Factores de Iniciación de Péptidos/química , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Soluciones , Difracción de Rayos XRESUMEN
Three protein factors ensure rapid and accurate initiation of translation in bacteria. Translation initiation factor IF2 is a ribosome-dependent GTPase, which is important for correct positioning of initiator tRNA on the 30S subunit as well as ribosomal subunit joining. The solution structure of the free C-terminal part of IF2 (IF2C, comprising domains IV to VI-2) was previously determined by small-angle X-ray scattering (SAXS) [Rasmussen, L. C., et al. (2008) Biochemistry 47, 5590-5598]. In this study, adding GDP or nonhydrolyzable GTP analogue GDPNP to the protein in solution caused structural changes in the protein, in agreement with recent data determined via isothermal titration calorimetry [Hauryliuk, V., et al. (2009) J. Mol. Biol. 394, 621-626]. The p(r) function indicated an elongated conformation supported by radii of gyration of 40.1 and 44.9 Å and maximum dimensions of ~125 and ~150 Å for IF2C with GDPNP and GDP, respectively. The SAXS data were used to model the structure of IF2C bound to either GDPNP or GDP. The structural transitions of IF2C upon GDPNP binding and following nucleotide hydrolysis support the concept of cofactor-dependent conformational switching rather than the classical model for GTPase activity.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Soluciones , Difracción de Rayos XRESUMEN
Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag. Our work is based on earlier observations where 3' deletions of the InfB gene displays strongly increased expression levels. We have constructed progressively shortened fragments of the InfB(1-471) gene and fused gene fragments to a gfp reporter gene. A 5-fold increase in GFP expression was seen for an optimal 21 nucleotide InfB(1-21) sequence compared to gfp independently. We defined the InfB(1-21) sequence as an expressivity tag. The tag was tested for improved expression of two biotechnological important proteins streptavidin and a single chain antibody (scFv). Expression of both streptavidin and scFv(L32) was improved as evaluated by SDS-PAGE. Calculation of folding energies in the translation initiation region gave higher free energies for gfp, L32 and streptavidin when linked to InfB(1-21) than independently. InfB(1-21) did however not improve the codon usage or codon adaptation index. The expressivity tag is an important addition to the box of tools available for optimizing heterologous protein expression.
Asunto(s)
Biotecnología/métodos , Clonación Molecular/métodos , Escherichia coli/genética , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Biosíntesis de Proteínas , Pliegue de Proteína , ARN Mensajero/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única , Solubilidad , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estadísticas no Paramétricas , TermodinámicaRESUMEN
Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule. Furthermore, the SAXS data revealed that E. coli IF2 in solution adopts a structure that is significantly different from the crystal structure of orthologous aIF5B from Methanobacterium thermoautotrophicum. This crystal structure constitutes the only atomic resolution structural knowledge of the full-length factor. Computer programs were applied to the SAXS data to provide an initial structural model for IF2 in solution. The low-resolution nature of SAXS prevents the elucidation of a complete and detailed structure, but the resulting model for C-terminal E. coli IF2 indicates important structural differences between the aIF5B crystal structure and IF2 in solution. The chalice-like structure with a highly exposed alpha-helical stretch observed for the aIF5B crystal structure was not found in the structural model of IF2 in solution, in which domain VI-2 is moved closer to the rest of the protein.
Asunto(s)
Escherichia coli/química , Escherichia coli/metabolismo , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Cristalografía por Rayos X , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Factor 2 Procariótico de Iniciación/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Initiation of protein synthesis in bacteria relies on the presence of three translation initiation factors, of which translation initiation factor IF1 is the smallest having a molecular weight of only 8.2kDa. In addition to its function in this highly dynamic process, the essential IF1 protein also functions as an RNA chaperone. Despite extensive research, the exact function of IF1 in translation initiation has not yet been determined, and the research in the function of the factor has in some areas been impeded by the lack of monoclonal antibodies specific for this protein. Several attempts to induce immune response in mice with wild-type IF1 for the production of antibodies have failed. We have now succeeded in producing monoclonal antibodies specific for IF1 by applying a new immunization strategy involving an antigen combination of IF1 coupled to glutathione S-transferase (GST) and a recombinant dimer of IF1. This resulted in the generation of 6 IgG, 2 IgM, and 1 IgA anti-IF1 antibodies, which can be used in ELISA screening and Western immunoblots. We also provide a mapping of the functional epitopes of the generated anti-IF1 monoclonal antibodies by screening the antibodies for binding to IF1 proteins mutated at single amino acid positions.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Factor 1 Procariótico de Iniciación/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Escherichia coli/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Factor 1 Procariótico de Iniciación/genética , Proteínas Recombinantes/biosíntesisRESUMEN
An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review.
RESUMEN
Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.
Asunto(s)
Chaperonas Moleculares/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN/metabolismo , Sustitución de Aminoácidos , Ensayo de Cambio de Movilidad Electroforética , Chaperonas Moleculares/genética , Factores de Iniciación de Péptidos/genética , Unión Proteica , Biosíntesis de Proteínas , ARN/química , ARN/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas de Escherichia coli/inmunología , Proteínas Fluorescentes Verdes/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Ribosómicas/inmunología , Animales , Escherichia coli/química , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Hibridomas , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process. Subsequent sections describe the structure, function, and interactions of the mRNA, the initiator tRNA, and the initiation factors IF1, IF2, and IF3. Finally, we provide an overview of mechanisms of regulation of the translation initiation event. Translation occurs on ribonucleoprotein complexes called ribosomes. The ribosome is composed of a large subunit and a small subunit that hold the activities of peptidyltransfer and decode the triplet code of the mRNA, respectively. Translation initiation is promoted by IF1, IF2, and IF3, which mediate base pairing of the initiator tRNA anticodon to the mRNA initiation codon located in the ribosomal P-site. The mechanism of translation initiation differs for canonical and leaderless mRNAs, since the latter is dependent on the relative level of the initiation factors. Regulation of translation occurs primarily in the initiation phase. Secondary structures at the mRNA ribosomal binding site (RBS) inhibit translation initiation. The accessibility of the RBS is regulated by temperature and binding of small metabolites, proteins, or antisense RNAs. The future challenge is to obtain atomic-resolution structures of complete initiation complexes in order to understand the mechanism of translation initiation in molecular detail.
Asunto(s)
Bacterias/genética , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Regulación Bacteriana de la Expresión Génica , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Ribosomes are extremely soluble ribonucleoprotein complexes. Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain. This enabled the isolation of 70S ribosomes with covalently bound target protein. Isolation of recombinant proteins from 70S ribosomes was achieved by specific proteolytic cleavage followed by efficient removal of ribosomes by centrifugation. By this procedure we isolated active green fluorescent protein, streptavidin (SA), and murine interleukin-6 (mIL-6). Approximately 500microg of each protein was isolated per gram cellular wet weight. By pull-down assays we demonstrate that SA covalently bound to the ribosome binds d-biotin. Ribosomal coupling is therefore suggested as a method for the investigation of protein interactions. The presented strategy is in particular efficient for the expression, purification, and investigation of proteins forming inclusion bodies in the E. coli cytoplasm.
Asunto(s)
Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Animales , Biotina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Plásmidos/genética , Plásmidos/metabolismo , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Ribosómicas/genética , Ribosomas/genéticaRESUMEN
Bacterial translation initiation factor IF2 is a multidomain protein that is an essential component of a system for ensuring that protein synthesis begins at the correct codon within a messenger RNA. Full-length IF2 from Escherichia coli and seven fragments of the protein were expressed, purified, and characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) methods. Interestingly, resonances of the 6 kD IF2N domain located at the extreme N terminus of IF2 can be clearly identified within the NMR spectra of the full-length 97-kD protein. (15)N NMR relaxation rate data indicate that (1) the IF2N domain is internally well ordered and tumbles in solution in a manner that is independent of the other domains of the IF2 protein, and (2) the IF2N domain is connected to the C-terminal regions of IF2 by a flexible linker. Chemical shifts of resonances within the isolated IF2N domain do not significantly differ from those of the corresponding residues within the context of the full-length 97-kD protein, indicating that IF2N is a structurally independent unit that does not strongly interact with other regions of IF2. CD and NMR data together provide evidence that Domains I-III of IF2 have unstructured and flexible regions as well as substantial helical content; CD data indicate that the helical content of these regions decreases significantly at temperatures above 35 degrees C. The features of structurally well-ordered N- and C-terminal domains connected by a flexible linker with significant helical content are reminiscent of another translation initiation factor, IF3.
Asunto(s)
Proteínas de Escherichia coli/química , Factor 2 Procariótico de Iniciación/química , Pliegue de Proteína , Secuencia de Aminoácidos , Dicroismo Circular , Clonación Molecular , Simulación por Computador , Secuencia Conservada , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/aislamiento & purificación , Factor 2 Procariótico de Iniciación/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones/química , TemperaturaRESUMEN
We have investigated different dialysis strategies for the refolding of recombinant streptavidin, and present a novel dialysis setup featuring gradual dilution dialysis and continuous protein feeding into a dialysis sack. A denaturing dialysis buffer is exchanged gradually by dilution with refolding buffer and it is demonstrated that the refolding yield can be increased from 45 to 75% by lowering the dilution rate. In addition, continuous feeding of protein to the dialysis sack increases the yield by 5 to 10%. The principle of gradual dilution dialysis is amenable to stringent regulation and we suggest it to be applied for other insoluble protein targets.
Asunto(s)
Pliegue de Proteína , Proteínas Recombinantes/química , Estreptavidina/química , Algoritmos , Cromatografía en Gel , Diálisis/instrumentación , Diálisis/métodos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Guanidina/química , Isopropil Tiogalactósido/farmacología , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Estreptavidina/biosíntesis , Estreptavidina/genéticaRESUMEN
Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E. coli often results in truncated protein forms. In many cases, this is a consequence of abundant codons in heterologous genes, which are decoded by rare tRNAs in E. coli-a combination that can be responsible for translational stalling and termination during protein biosynthesis. Other complications may originate from potential initiation codons and ribosomal binding sites present inside the open reading frame of the target gene or from other less well defined phenomena such as mRNA instability. Separation of full-length protein from truncated forms is a serious chromatographic problem that can be solved in the expression step. We have investigated the heterologous expression and purification of two translation initiation factors from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus. Expression in E. coli was optimised to avoid truncated forms completely by complementation with the plasmids pSJS1244, pRIG, pCODON+ and pLysSR.A.R.E harbouring and expressing genes encoding rare tRNAs corresponding to the codons AGA, AGG, AUA, CUA, GGA, AAG and CCC. Two expression strains, C41(DE3) and C43(DE3) were found highly advantageous when combined with rare tRNA encoding plasmids as compared to BL21(DE3). We have also investigated the effects of site directed mutagenesis on rare lysine encoding AAG doublets as well as two methionine residues preceded by potential ribosomal binding sites. The expression approach presented here has enabled us to purify gram quantities of full-length protein by one step of ion-exchange chromatography and is generally applicable to many other heterologously expressed thermostable proteins.
Asunto(s)
Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ADN Recombinante , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2-50 were found to form a compact subdomain containing three short beta-strands and three alpha-helices, folded to form a betaalphaalphabetabetaalpha motif with the three helices packed on the same side of a small twisted beta-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51-97 are less conserved and do not appear to form a regular structure, whereas residues 98-157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2.
Asunto(s)
Proteínas de Escherichia coli/química , Factor 2 Procariótico de Iniciación/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dicroismo Circular , Secuencia Conservada , Mapeo Epitopo , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Pliegue de ProteínaRESUMEN
The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dicroismo Circular , Clonación Molecular , Frío , Escherichia coli/genética , GTP Fosfohidrolasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos , Factor 2 Procariótico de Iniciación/genética , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Mapeo RestrictivoRESUMEN
Recombinant streptavidin is extremely difficult to express at high levels in the cytoplasm of Escherichia coli without the formation of inclusion bodies. Fusing a solubility enhancing partner to an aggregation prone protein is a widely used tool to circumvent inclusion body formation. Here, we use streptavidin as a target protein to test the properties of N-terminal fragments of translation initiation factor IF2 from E. coli as a solubility partner. Domain I (residue 1-158) of IF2 is superior to the well-established solubility partners maltose-binding protein (MBP) and NusA for soluble expression of active streptavidin. The number of active streptavidin molecules isolated by chromatography is increased threefold when domain I is used as solubility partner as compared to MBP or NusA. The relatively small size, high expressivity, and extreme solubility make domain I of IF2 an ideal partner for streptavidin and may also prevent other recombinant proteins such as ScFv antibodies from being expressed as insoluble aggregates in the cytoplasm of E. coli.
Asunto(s)
Factor 2 Procariótico de Iniciación/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Estreptavidina/biosíntesis , Estreptavidina/química , Unión Competitiva , Biotina/metabolismo , Proteínas Portadoras/química , Cartilla de ADN/genética , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Proteínas de Unión a Maltosa , Factores de Elongación de Péptidos/química , Factor 2 Procariótico de Iniciación/biosíntesis , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Estreptavidina/genética , Estreptavidina/metabolismo , Factores de Transcripción/química , Factores de Elongación TranscripcionalRESUMEN
BACKGROUND: The gene infB encodes the prokaryotic translation initiation factor IF2, a central macromolecular component in the formation of the ribosomal 70S initiation complex. In Escherichia coli, infB encodes three forms of IF2: IF2alpha, IF2beta and IF2gamma. The expression of IF2beta and IF2gamma is a tandem translation from intact infB mRNA and not merely a translation of post-transcriptionally truncated mRNA. The molecular mechanism responsible for the ribosomal recognition of the two intracistronic translation initiation sites in E. coli infB is not well characterized. RESULTS: We found three different forms of IF2 in Enterobacter cloacae, Klebsiella oxytoca, Salmonella enterica, Salmonella typhimurium, and two different forms in Proteus vulgaris. We identified the intracistronic translation initiation sites of the mRNA by isolation and N-terminal sequencing of the shorter isoforms of IF2 in S. enterica and S. typhimurium. A further search in the readily available public sequence databases revealed that infB from Yersinia pestis also contains an intracistronic in-frame initiation site used for the translation of IF2beta. The base composition in a part of the 5' end of the DNA coding strand of the enterobacterial infB gene shows a strong preference for adenine (A) over thymine (T) with a maximum ratio of A-to-T around the intracistronic initiation sites. We demonstrate that the mRNA has an open structure around the ribosomal binding region. CONCLUSION: Efficient intracistronic translation initiation of the infB gene is suggested to require an mRNA with this special base composition that results in an open, single-stranded structure at the ribosomal binding region.
Asunto(s)
Enterobacteriaceae/genética , Conformación de Ácido Nucleico , Factor 2 Procariótico de Iniciación/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Enterobacter/genética , Enterobacteriaceae/metabolismo , Genes Bacterianos , Klebsiella oxytoca/genética , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica Traduccional , Isoformas de Proteínas/genética , Proteus vulgaris/genética , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Salmonella enterica/genética , Alineación de SecuenciaRESUMEN
The sequence of infB, encoding the prokaryotic translation-initiation factor 2 (IF2), was determined in eight strains of Streptococcus agalactiae (group B streptococcus) and an alignment revealed limited intraspecies diversity within S. agalactiae. The amino acid sequence of IF2 from S. agalactiae and from related species were aligned and revealed an interspecies conserved central and C-terminal part, and an N-terminal part that is highly variable in length and amino acid sequence. The diversity and relationships in a collection of 58 genetically distinct strains of S. agalactiae were evaluated by comparing a partial sequence of infB. A total of six alleles were detected for the region of infB analysed. The alleles correlated with the separation of the same strains of S. agalactiae into major evolutionary lineages, as shown in previous work. The partial sequences of infB were furthermore used in phylogenetic analyses of species closely related to S. agalactiae, yielding an evolutionary tree which had a topology similar to a tree constructed using 16S rRNA sequences from the same species.