RESUMEN
The cauliflower mosaic virus transactivator, TAV, controls translation reinitiation of major open reading frames on polycistronic RNA. We show here that TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 in vitro and in vivo. TAV physically interacts with eIF3 and the 60S ribosomal subunit. Two proteins mediating these interactions were identified: eIF3g and 60S ribosomal protein L24. Transient expression of eIF3g and L24 in plant protoplasts strongly affects TAV-mediated reinitiation activity. We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to polysomes, allowing translation of polycistronic mRNAs by reinitiation, overcoming the normal cell barriers to this process.
Asunto(s)
Brassica/genética , Brassica/virología , Caulimovirus/genética , Regulación de la Expresión Génica de las Plantas , Biosíntesis de Proteínas , Transactivadores/genética , Transactivadores/metabolismo , Secuencia de Aminoácidos , Caulimovirus/fisiología , Clonación Molecular , Secuencia Conservada , Factor 3 de Iniciación Eucariótica , Genes de Plantas , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Factores de Iniciación de Péptidos/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transactivadores/química , Transcripción Genética , Triticum/genéticaAsunto(s)
Caulimovirus/genética , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Brassica/virología , Caulimovirus/ultraestructura , Codón/genética , Genes Virales , Sistemas de Lectura Abierta , Protoplastos/fisiología , Transfección , Proteínas Estructurales Virales/genéticaRESUMEN
The pathways of scanning ribosome migration controlled by the cauliflower mosaic virus 35 S RNA leader were investigated in vitro and in vivo. This long (600 nucleotides) leader contains several short open reading frames (sORFs) and folds into an extended hairpin structure with three main stable stem sections. Translation initiation downstream of the leader is cap-dependent and occurs via ribosomal shunt under the control of two cis elements, a short open reading frame A (sORF A) followed by stem section 1. Here we show that a second similar configuration comprising sORF B followed by stem section 2 also allows shunting. The efficiency of the secondary shunt was greatly increased when stem section 1 was destabilized. In addition, we present evidence that a significant fraction of reinitiation-competent ribosomes that escape both shunt events migrate linearly via the structured central region but are intercepted by internal AUG start codons. Thus, expression downstream of the 35 S RNA leader is largely controlled by its multiple sORFs.
Asunto(s)
Regiones no Traducidas 5'/genética , Caulimovirus/genética , Sistemas de Lectura Abierta , Ribosomas/genética , Secuencia de Bases , Codón Iniciador , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/genéticaRESUMEN
The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning-reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5'-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5'-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of reinitiation.
Asunto(s)
Caulimovirus/genética , Sistemas de Lectura Abierta , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , ARN Lider Empalmado/genética , ARN Viral/genética , Ribosomas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Libre de Células , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Conejos , Reticulocitos/metabolismo , Ribosomas/genética , Semillas , Transcripción Genética , TriticumRESUMEN
Prokaryotic and eukaryotic in vitro translation systems have recently become the focus of increasing interest for tackling fundamental problems in biochemistry. Cell-free systems can now be used to study the in vitro assembly of membrane proteins and viral particles, rapidly produce and analyze protein mutants, and enlarge the genetic code by incorporating unnatural amino acids. Using in vitro translation systems, display techniques of great potential have been developed for protein selection and evolution. Furthermore, progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Animales , Reactores Biológicos , Biotecnología , Sistema Libre de Células/metabolismo , Metabolismo Energético , Humanos , Mutación , Biblioteca de Péptidos , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Virus/genética , Virus/crecimiento & desarrollo , Virus/metabolismoRESUMEN
A wheat germ cell-free system was used to study details of ribosome shunting promoted by the cauliflower mosaic virus 35 S RNA leader. By testing a dicistronic construct with the leader placed between two coding regions, we confirmed that the 35 S RNA leader does not include an internal ribosome entry site of the type observed with picornavirus RNAs. A reporter gene fused to the leader was shown to be expressed by ribosomes that had followed the bypass route (shunted) and, with lower efficiency, by ribosomes that had scanned through the whole region. Stem section 1, the most stable of the three stem sections of the leader, was shown to be an important structural element for shunting. Mutations that abolished formation of this stem section drastically reduced reporter gene expression, whereas complementary mutations that restored stem section 1 also restored shunting. A micro-leader capable of shunting consisting of stem section 1 and flanking sequences could be defined. A small open reading frame preceding stem section 1 enhances shunting.
Asunto(s)
Caulimovirus/genética , Ribosomas/metabolismo , Composición de Base , Secuencia de Bases , Sistema Libre de Células , Cloranfenicol O-Acetiltransferasa/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genéticaRESUMEN
To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffing, and not net formation, is the crucial yield-limiting step. The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation. None of these factors significantly influenced total protein synthesis. In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 micrograms/ml in only 15 min) could be recovered from immobilized antigen.
Asunto(s)
Formación de Anticuerpos/genética , Isomerasas/genética , Isomerasas/metabolismo , Chaperonas Moleculares/metabolismo , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Anticuerpos/genética , Western Blotting , Escherichia coli/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Proteína Disulfuro Isomerasas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaAsunto(s)
Proteínas Bacterianas/biosíntesis , Escherichia coli/genética , Organofosfatos/metabolismo , Biosíntesis de Proteínas , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Metabolismo Energético , Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Fosfoenolpiruvato/metabolismo , Piruvato Quinasa/metabolismoRESUMEN
In order to test the enhancing effect of the 3'-terminal untranslated region (3'-UTR) of tobacco mosaic virus (TMV) RNA on protein synthesis in vitro we used a chimeric mRNA construct containing TMV 5'-UTR (omega) and firefly luciferase mRNA. The addition of the TMV 3'-UTR to the chimeric mRNA construct results in a more than 3-fold stimulation of the synthesis of the functionally active protein in the wheat germ cell-free translation system. We have demonstrated that the proper length of the TMV 3'-terminal part is important for efficient translation; elongation of the TMV tail by 160 vector-derived nucleotides fully abolishes the stimulation effect of the TMV 3'-UTR in vitro.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/farmacología , ARN Viral/farmacología , Virus del Mosaico del Tabaco/genética , Secuencia de Bases , Sistema Libre de Células , ARN Polimerasas Dirigidas por ADN/genética , Estabilidad de Medicamentos , Luciferasas/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN/farmacología , Proteínas ViralesRESUMEN
In order to understand the role of the 3'-terminal untranslated region (3'-UTR) of alfalfa mosaic virus (AlMV) RNA 4 in viral RNA translation we have constructed the RNA derivatives differing in the length of their 3'-terminal portions and expressed them in a wheat germ extract. The result shows that the removal of the 3'-UTR from AlMV RNA 4 causes a lagged RNA translation in the cell-free system as compared with the translation of the full length RNA 4, thus suggesting the involvement of the 3'-UTR in the translation initiation pathway.
Asunto(s)
Virus del Mosaico/genética , Biosíntesis de Proteínas , ARN Viral/química , ARN Viral/metabolismo , Sistema Libre de Células , Cinética , ARN Mensajero/química , ARN Mensajero/metabolismo , Relación Estructura-Actividad , TriticumRESUMEN
A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer [including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)] through the reaction mixture and a continuous removal of a polypeptide product. Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested. In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours. With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively. With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.
Asunto(s)
Escherichia coli/metabolismo , Biosíntesis de Péptidos , Plantas/metabolismo , Biosíntesis de Proteínas , Bacteriófagos/genética , Calcitonina/biosíntesis , Calcitonina/genética , Cápside/biosíntesis , Cápside/genética , Electroforesis , Cinética , Virus del Mosaico/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Ribosomas/metabolismo , Moldes Genéticos , TriticumRESUMEN
The release of deacylated tRNA from the ribosome as a result of translocation has been studied. Translating ribosomes prepared with poly(U)-S-S-Sepharose columns have been used. It has been shown that deacylated tRNA released from the ribosomal P site as a result of translocation rebinds with the vacated A site. Consistent with the known properties of the A site of the ribosome, this interaction is reversible, Mg2+-dependent, codon-specific and is inhibited by the antibiotic tetracycline. It has been concluded that the proposed three-site model of the ribosomal elongation cycle (Rheinberger and Nierhaus (1983) Proc. Natl. Acad. Sci. USA 80, 4213-4217) is not sound: the experimentally observed 'retention' of the deacylated tRNA on the ribosome after translocation can be explained by a codon-dependent rebinding to the A site, rather than by its transition to the 'E site', i.e., in terms of the classical two-site model.
Asunto(s)
Extensión de la Cadena Peptídica de Translación , Ribosomas/fisiología , Modelos Biológicos , Biosíntesis de Proteínas , ARN de Transferencia de Fenilalanina/genética , Translocación GenéticaRESUMEN
MS2 phage RNA-directed synthesis of an N-terminal polypeptide of the phage coat protein on Escherichia coli 70 S ribosomes was initiated in a cell-free system with the N-dinitrophenyl derivative of methionyl-tRNAFMet) and performed in the absence of tyrosine, lysine, cysteine and methionine. As a result, the translating ribosomes carried peptides up to 42 amino acid residues in length with the dinitrophenyl hapten at the N-ends. Using the immune electron microscopy technique the positions of the nascent peptide N-ends on the 70 S ribosomes have been visualized. It has been found that (i) the N-ends of nascent peptides of these lengths are accessible to antibodies, (ii) the exit site of a nascent peptide is the pocket between the base of the central protuberance and the L1 ridge on the 50 S subunit, i.e. presumably its peptidyl transferase center, and (iii) the further pathway of a nascent peptide seems to proceed along the groove on the external surface of the 50 S subunit.