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1.
Viruses ; 10(12)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30558257

RESUMO

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation.


Assuntos
Caulimovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Caulimovirus/genética , Núcleo Celular/química , Núcleo Celular/virologia , Citoplasma/química , Citoplasma/virologia , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Fases de Leitura Aberta , Doenças das Plantas/virologia , Fator de Iniciação 3 em Procariotos/genética , Interferência de RNA , Tabaco/citologia , Tabaco/virologia , Transativadores/genética , Proteínas Virais/genética
2.
MBio ; 8(3)2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28611250

RESUMO

The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry.IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR-rsrA, we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons.


Assuntos
Códon de Iniciação , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , Fator de Iniciação 3 em Procariotos/metabolismo , Fator sigma/metabolismo , Streptomyces/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Óperon , Fator de Iniciação 3 em Procariotos/genética , Regulon , Fator sigma/química , Streptomyces/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Plant Physiol ; 172(2): 1117-1130, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27535792

RESUMO

Chloroplast development requires the coordinated expressions of nuclear and chloroplast genomes, and both anterograde and retrograde signals exist and work together to facilitate this coordination. We have utilized the Arabidopsis yellow variegated (var2) mutant as a tool to dissect the genetic regulatory network of chloroplast development. Here, we report the isolation of a new (to our knowledge) var2 genetic suppressor locus, SUPPRESSOR OF VARIEGATION9 (SVR9). SVR9 encodes a chloroplast-localized prokaryotic type translation initiation factor 3 (IF3). svr9-1 mutant can be fully rescued by the Escherichia coli IF3 infC, suggesting that SVR9 functions as a bona fide IF3 in the chloroplast. Genetic and molecular evidence indicate that SVR9 and its close homolog SVR9-LIKE1 (SVR9L1) are functionally interchangeable and their combined activities are essential for chloroplast development and plant survival. Interestingly, we found that SVR9 and SVR9L1 are also involved in normal leaf development. Abnormalities in leaf anatomy, cotyledon venation patterns, and leaf margin development were identified in svr9-1 and mutants that are homozygous for svr9-1 and heterozygous for svr9l1-1 (svr9-1 svr9l1-1/+). Meanwhile, as indicated by the auxin response reporter DR5:GUS, auxin homeostasis was disturbed in svr9-1, svr9-1 svr9l1-1/+, and plants treated with inhibitors of chloroplast translation. Genetic analysis established that SVR9/SVR9L1-mediated leaf margin development is dependent on CUP-SHAPED COTYLEDON2 activities and is independent of their roles in chloroplast development. Together, our findings provide direct evidence that chloroplast IF3s are essential for chloroplast development and can also regulate leaf development.


Assuntos
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Folhas de Planta/genética , Fator de Iniciação 3 em Procariotos/genética , Sequência de Aminoácidos , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Microscopia Confocal , Mutação , Filogenia , Folhas de Planta/anatomia & histologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Fator de Iniciação 3 em Procariotos/classificação , Fator de Iniciação 3 em Procariotos/metabolismo , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
4.
Med Princ Pract ; 25(5): 461-5, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27333287

RESUMO

OBJECTIVE: To investigate single nucleotide polymorphisms in the eukaryotic translation initiation factor 3a (eIF3a) gene and the risk for gastric cancer within the Chinese population. SUBJECTS AND METHODS: A total of 322 patients with gastric cancer were selected as the patient group and 340 non-gastric cancer patients were selected as the control group using the case-control method. Polymerase chain reaction-sequence-specific primer technology was leveraged to genotype the rs77382849 single nucleotide polymorphism in the eIF3a gene. The demographic characteristics of the study population and other exposures to risk factors were collected. Unconditional logistic regression analysis was performed to determine the association between the risk factors and gastric cancer. RESULTS: A higher frequency of the eIF3a rs77382849 GG homozygote genotype was observed in the gastric cancer patients compared with the controls (63.98 vs. 54.41%, p < 0.05). After adjustment of exposure risks, such as age, gender, smoking, and drinking, the rs77382849 single nucleotide polymorphism was still associated with susceptibility to gastric cancer. When the eIF3a rs77382849 GG homozygote genotype was used as the reference group, the GA genotype (GA vs. GG: OR = 0.545, 95% CI: 0.386-0.769, p = 0.001) and AA genotype (AA vs. GG: OR = 0.245, 95% CI: 0.072-0.836, p = 0.025) were both correlated with a significantly decreased risk for gastric cancer development. CONCLUSION: An association between eIF3a rs77382849 polymorphism and susceptibility to gastric cancer was observed in these Chinese patients.


Assuntos
Fator de Iniciação 3 em Procariotos/genética , Neoplasias Gástricas/genética , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , China , Feminino , Predisposição Genética para Doença , Genótipo , Comportamentos Relacionados com a Saúde , Humanos , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Fatores Socioeconômicos
5.
Fish Physiol Biochem ; 41(3): 695-704, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25743365

RESUMO

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has been used extensively for studying gene expression in diverse organisms including fish. In this study, with an aim to identify reliable reference genes for qRT-PCR in red drum (Sciaenops ocellatus), an economic fish species, we determined the expression stability of seven housekeeping genes in healthy and bacterium-infected red drum. Each of the selected candidate genes was amplified by qRT-PCR from the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of red drum infected with or without a bacterial pathogen for 12 and 48 h. The mRNA levels of the genes were analyzed with the geNorm and NormFinder algorithms. The results showed that in the absence of bacterial infection, translation initiation factor 3, NADH dehydrogenase 1, and QM-like protein may be used together as internal references across the eight examined tissues. Bacterial infection caused variations in the rankings of the most stable genes in a tissue-dependent manner. For all tissues, two genes sufficed for reliable normalization at both 12 and 48 h post-infection. However, the optimal gene pairs differed among tissues and, for four of the examined eight tissues, between infection points. These results indicate that when studying gene expression in red drum under conditions of bacterial infection, the optimal reference genes should be selected on the basis of tissue type and, for accurate normalization, infection stage.


Assuntos
Regulação da Expressão Gênica/genética , Genes Essenciais/genética , Perciformes/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Encéfalo/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Brânquias/metabolismo , Músculo Esquelético/metabolismo , Fator de Iniciação 3 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Vísceras/metabolismo
6.
J Mol Biol ; 427(9): 1801-18, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25308340

RESUMO

Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.


Assuntos
Fator de Iniciação 2 em Procariotos/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/metabolismo , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Conformação Molecular , Fator de Iniciação 2 em Procariotos/química , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/química , Fator de Iniciação 3 em Procariotos/genética , RNA Mensageiro/genética , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química
7.
Biochimie ; 100: 132-40, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23954798

RESUMO

The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , Sequência de Aminoácidos , Evolução Biológica , Transporte de Elétrons/genética , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 2 em Procariotos/metabolismo , Fator de Iniciação 3 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
8.
Proc Natl Acad Sci U S A ; 110(40): 16253-8, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24048028

RESUMO

Light-harvesting antennae are critical for collecting energy from sunlight and providing it to photosynthetic reaction centers. Their abundance and composition are tightly regulated to maintain efficient photosynthesis in changing light conditions. Many cyanobacteria alter their light-harvesting antennae in response to changes in ambient light-color conditions through the process of chromatic acclimation. The control of green light induction (Cgi) pathway is a light-color-sensing system that controls the expression of photosynthetic genes during chromatic acclimation, and while some evidence suggests that it operates via transcription attenuation, the components of this pathway have not been identified. We provide evidence that translation initiation factor 3 (IF3), an essential component of the prokaryotic translation initiation machinery that binds the 30S subunit and blocks premature association with the 50S subunit, is part of the control of green light induction pathway. Light regulation of gene expression has not been previously described for any translation initiation factor. Surprisingly, deletion of the IF3-encoding gene infCa was not lethal in the filamentous cyanobacterium Fremyella diplosiphon, and its genome was found to contain a second, redundant, highly divergent infC gene which, when deleted, had no effect on photosynthetic gene expression. Either gene could complement an Escherichia coli infC mutant and thus both encode bona fide IF3s. Analysis of prokaryotic and eukaryotic genome databases established that multiple infC genes are present in the genomes of diverse groups of bacteria and land plants, most of which do not undergo chromatic acclimation. This suggests that IF3 may have repeatedly evolved important roles in the regulation of gene expression in both prokaryotes and eukaryotes.


Assuntos
Cianobactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Transdução de Sinal Luminoso/fisiologia , Luz , Família Multigênica/genética , Fotossíntese/fisiologia , Fator de Iniciação 3 em Procariotos/metabolismo , Sequência de Bases , Biologia Computacional , Primers do DNA/genética , Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Transdução de Sinal Luminoso/genética , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Fator de Iniciação 3 em Procariotos/genética , Análise de Sequência de DNA , Especificidade da Espécie
9.
Cell Stress Chaperones ; 17(2): 145-56, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22002548

RESUMO

The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70-peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a 'pull-down' assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be up-regulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.


Assuntos
Neoplasias da Mama/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Biomarcadores , Biomarcadores Tumorais , Linhagem Celular Tumoral , Fator de Iniciação 3 em Eucariotos/genética , Feminino , Imunofluorescência , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Espectrometria de Massas , Análise em Microsséries , Complexos Multiproteicos/química , Biblioteca de Peptídeos , Fator de Iniciação 3 em Procariotos/genética , Regulação para Cima
10.
FEMS Microbiol Lett ; 313(2): 141-7, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21054500

RESUMO

Using a specialized ribosome system, previous studies have identified G791 in Escherichia coli 16S rRNA as an invariant and essential residue for ribosome function. To investigate the functional role of G791, we searched for multicopy suppressors that partially restored the protein synthesis ability of mutant ribosomes bearing a G to U substitution at position 791 (U791 ribosomes). Analyses of isolated multicopy suppressors showed that overexpression of initiation factor 1 (IF1) enhanced the protein synthesis ability of U791 ribosomes. In contrast, overexpression of initiation factor 2 (IF2) or IF3 did not enhance the protein synthesis ability of wild-type or U791 ribosomes, and overexpression of IF1 did not affect the function of wild-type or mutant ribosomes bearing nucleotide substitutions in other regions of 16S rRNA. Analyses of sucrose gradient profiles of ribosomes showed that overexpression of IF1 marginally enhanced the subunit association of U791 ribosomes and indicated lower binding affinity of U791 ribosomes to IF1. Our findings suggest the involvement of IF1 in the restoration of the P-site function that was impaired by a nucleotide substitution at residue G791.


Assuntos
Escherichia coli/metabolismo , Fator de Iniciação 1 em Procariotos/metabolismo , Biossíntese de Proteínas , RNA Ribossômico 16S/metabolismo , Ribossomos/metabolismo , Escherichia coli/genética , Mutação Puntual , Fator de Iniciação 1 em Procariotos/genética , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/genética , RNA Ribossômico 16S/genética , Subunidades Ribossômicas/metabolismo , Ribossomos/genética , Supressão Genética
11.
Folia Microbiol (Praha) ; 55(6): 607-13, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21253907

RESUMO

PCR and SYBR Green I real-time PCR techniques were applied to the rapid detection of Vibrio metschnikovii by designing primers based on infC (initiation factor 3) gene sequence. The specificity, sensitivity, and practical applications of the methods have been also analyzed. The methods showed high detecting specificity with no cross amplifications of other closely related and nonrelated species; they provide a simple and rapid tool for V. metschnikovii detection with high sensitivity and specificity.


Assuntos
Técnicas Bacteriológicas/métodos , Reação em Cadeia da Polimerase/métodos , Vibrio/isolamento & purificação , Microbiologia da Água , Primers do DNA/genética , Compostos Orgânicos/metabolismo , Fator de Iniciação 3 em Procariotos/genética , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Fatores de Tempo
12.
Biochemistry ; 48(22): 4699-706, 2009 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-19366171

RESUMO

Addition of an Escherichia coli 50S subunit (50S(Cy5)) containing a Cy5-labeled L11 N-terminal domain (L11-NTD) within the GTPase-associated center (GAC) to an E. coli 30S initiation complex (30SIC(Cy3)) containing Cy3-labeled initiation factor 2 complexed with GTP leads to rapid development of a FRET signal during formation of the 70S initiation complex (70SIC). Initiation factor 2 (IF2) and elongation factor G (EF-G) induce similar changes in ribosome structure. Here we show that such similarities are maintained on a dynamic level as well. Thus, movement of IF2 toward L11-NTD after initial 70S ribosome formation follows GTP hydrolysis and precedes P(i) release, paralleling movement of EF-G following its binding to the ribosome [Seo, H., et al. (2006) Biochemistry 45, 2504-2514], and in both cases, the rate of such movement is slowed if GTP hydrolysis is prevented. The 30SIC(Cy3):50S(Cy5) FRET signal also provides a sensitive probe of the ability of initiation factor 3 to discriminate between a canonical and a noncanonical initiation codon during 70SIC formation. We employ Bacillus stearothermophilus IF2 as a substitute for E. coli IF2 to take advantage of the higher stability of the complexes it forms with E. coli ribosomes. While Bst-IF2 is fully functional in formation of E. coli 70SIC, relative reactivities toward dipeptide formation of 70SICs formed with the two IF2s suggest that the Bst-IF2.GDP complex is more difficult to displace from the GAC than the E. coli IF2.GDP complex.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 2 em Procariotos/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , GTP Fosfo-Hidrolases/química , Geobacillus stearothermophilus , Hidrólise , Luz , Fator de Iniciação 2 em Procariotos/química , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/química , Fator de Iniciação 3 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Ribossômicas/biossíntese , Ribossomos/enzimologia , Espalhamento de Radiação
13.
Biochemistry ; 48(15): 3269-78, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19239245

RESUMO

Mitochondrial translational initiation factor 3 (IF3(mt)) is a 29 kDa protein that has N- and C-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domains are preceded and followed by short extensions. No information is currently available on the specific residues in IF3(mt) important for its activity. On the basis of homology models of IF3(mt), mutations were designed in the N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutation of residues 170-171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss of activity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However, these mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with K(d) values similar to that of the wild-type factor. These mutations appear to lead to a factor defective in the ability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process. Other mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or no effect on the ability of IF3(mt) to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3(mt) to reduce the level of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3(mt) plays an active role in initiation of translation.


Assuntos
Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Biossíntese de Proteínas , Animais , Bovinos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fatores de Iniciação em Eucariotos/fisiologia , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Humanos , Camundongos , Proteínas Mitocondriais/fisiologia , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fator de Iniciação 3 em Procariotos/química , Fator de Iniciação 3 em Procariotos/genética , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
14.
Immunobiology ; 214(6): 467-74, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19150742

RESUMO

Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.


Assuntos
Infecções por Alphavirus/imunologia , Fatores de Iniciação em Eucariotos/imunologia , Fator de Iniciação 3 em Procariotos/imunologia , Vírus da Floresta de Semliki/imunologia , Vacinação , Infecções por Alphavirus/prevenção & controle , Animais , Brucella abortus/genética , Fatores de Iniciação em Eucariotos/genética , Engenharia Genética , Imunidade Ativa/genética , Camundongos , Camundongos Endogâmicos BALB C , Fator de Iniciação 3 em Procariotos/genética , Recombinação Genética , Vírus da Floresta de Semliki/patogenicidade , Virulência
15.
Immunobiology ; 214(2): 113-20, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19167989

RESUMO

The infC gene of Brucella abortus encoding the translation initiation factor 3 (IF3) was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence analysis predicted a product with 74-80% identity with the IF3 proteins from Mesorhizobium loti, Sinorhizobium meliloti, Aurantimona sp. and Mesorhizobium sp. This protein also show 54% amino acid sequence identity with the E. coli IF3, sharing most of the residues which were described as responsible for the biological activity of this protein. Since we have previously reported the immunoprotective capacity of this Brucella protein, we stimulated lymphoid cells from animals immunized with purified recombinant Brucella IF3 protein "in vitro" with this antigen. The lymphocytes were able to mount a strong proliferative response with concomitant production of gamma interferon, but without the secretion of either IL-4 or antibodies. Thus, immunization with the Brucella recombinant IF3 protein promotes a TH-1 polarized response, allowing us to propose it as a promising candidate antigen for the development of subunit vaccines against Brucella.


Assuntos
Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Fator de Iniciação 3 em Procariotos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Vacina contra Brucelose/genética , Células Cultivadas , Clonagem Molecular , Feminino , Expressão Gênica , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fator de Iniciação 3 em Procariotos/genética , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia
16.
J Mol Biol ; 384(4): 929-40, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-18930736

RESUMO

Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3(mt)) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3(mt) with and without the connecting linker were prepared. The K(d) values for the binding of full-length IF3(mt) and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3(mt). This observation indicates that the N-domain of IF3(mt) has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3(mt) for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3(mt) in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3(mt) reduces the binding of fMet-tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3(mt) promotes the formation of a binary complex between IF2(mt) and fMet-tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Animais , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Iniciação em Eucariotos/genética , Cinética , Fator de Iniciação 3 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Ribossômicas Menores/metabolismo
17.
J Mol Biol ; 383(5): 937-44, 2008 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-18805426

RESUMO

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3(Y75N) protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed.


Assuntos
Escherichia coli/metabolismo , Mutação/genética , Fator de Iniciação 3 em Procariotos/química , Fator de Iniciação 3 em Procariotos/genética , Biossíntese de Proteínas , Regiões 5' não Traduzidas/metabolismo , Alelos , Sequência de Aminoácidos , Teste de Complementação Genética , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo
18.
Biochemistry ; 47(34): 8828-39, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18672904

RESUMO

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mycobacterium smegmatis/genética , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fator de Iniciação 1 em Procariotos/genética , Fator de Iniciação 1 em Procariotos/metabolismo , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 2 em Procariotos/metabolismo , Fator de Iniciação 3 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/metabolismo , Fatores de Iniciação em Procariotos , Ligação Proteica , RNA de Transferência de Fenilalanina/metabolismo , Subunidades Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Homologia de Sequência de Aminoácidos
19.
J Mol Biol ; 380(3): 451-64, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18565340

RESUMO

Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation. Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood. We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA-minigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters tRNA(Arg4) as a short-chain peptidyl-tRNA(Arg4) in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-tRNA(Arg4) from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on lambdaimmP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Alelos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos , Fator de Iniciação 3 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/fisiologia , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
20.
Mol Microbiol ; 67(5): 1012-26, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18221266

RESUMO

Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV-geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA - formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site - are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 2 em Procariotos/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , Bacteriófago M13/crescimento & desenvolvimento , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 3 em Procariotos/genética , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética , Ribossomos/metabolismo
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