RESUMEN
Translational reading gaps occur when genetic information encoded in mRNA is not translated during the normal course of protein synthesis. This phenomenon has been observed thus far only in prokaryotes and is a mechanism for extending the reading frame by circumventing the normal stop codon. Reading frames of proteins may also be extended by suppression of the stop codon mediated by a suppressor tRNA. The rabbit beta-globin read-through protein, the only known, naturally occurring read-through protein in eukaryotes, was sequenced by ion trap mass spectrometry to determine how the reading frame is extended. Seven different proteolytic peptide fragments decoded by the same sequence that spans the UGA stop codon of rabbit beta-globin mRNA were detected. Three of these peptides contain translational reading gaps of one to three amino acids that correspond to the UGA stop codon site and/or one or two of the immediate downstream codons. To our knowledge, this is the first reported example of the occurrence of reading gaps in protein synthesis in eukaryotes. This event is unique in that it is associated with bypasses involving staggered lengths of untranslated information. Four of the seven peptides contain serine, tryptophan, cysteine, and arginine decoded by UGA and thus arise by suppression. Serine is donated by selenocysteine tRNA, and it, like the other tRNAs, has previously been shown to suppress UGA in vitro in mammals, but not in vivo.
Asunto(s)
Codón de Terminación/química , Globinas/genética , Biosíntesis de Proteínas , Sistemas de Lectura/genética , Supresión Genética , Secuencia de Aminoácidos , Animales , Arginina/genética , Codón de Terminación/genética , Cisteína/genética , Globinas/biosíntesis , Globinas/química , Datos de Secuencia Molecular , Terminación de la Cadena Péptídica Traduccional/genética , Fragmentos de Péptidos/genética , Conejos , Serina/genética , Triptófano/genéticaRESUMEN
We reported previously that the selenium status of rats influences both the steady-state levels and distributions of two selenocysteine tRNA isoacceptors and that these isoacceptors differ by a single methyl group attached to the ribosyl moiety at position 34. In this study, we demonstrate that repletion of selenium-deficient rats results in a gradual, tissue-dependent shift in the distribution of these isoacceptors. Rats fed a selenium-deficient diet possess a greater abundance of the species unmethylated on the ribosyl moiety at position 34 compared to the form methylated at this position. A redistribution of the Sec-tRNA isoacceptors occurred in tissues of selenium-supplemented rats whereby the unmethylated form gradually shifted toward the methylated form. This was true in each of four tissues examined, muscle, kidney, liver and heart, although the rate of redistribution was tissue-specific. Muscle manifested a predominance of two minor serine isoacceptors under conditions of extreme selenium-deficiency which also appeared to respond to selenium. Ribosomal binding studies revealed that one of the two additional isoacceptors decodes the serine codeword, AGU, and the second decodes the serine codeword, UCU. Interestingly, muscle and heart were the slower tissues to return to a 'selenium adequate' tRNA distribution pattern.
Asunto(s)
ARN de Transferencia Aminoácido-Específico/metabolismo , Selenio/deficiencia , Selenio/metabolismo , Animales , Cromatografía por Intercambio Iónico , Codón/genética , Dieta , Riñón/metabolismo , Hígado/metabolismo , Masculino , Músculos/metabolismo , Miocardio/metabolismo , Especificidad de Órganos , Proteínas/metabolismo , ARN de Transferencia de Serina/metabolismo , Ratas , Ratas Sprague-Dawley , Ribosomas/metabolismo , Selenio/administración & dosificación , SelenoproteínasRESUMEN
Selenocysteine (Sec) tRNAs serve as carrier molecules for the biosynthesis of Sec from serine and to donate Sec to protein in response to specific UGA codons. In this study, we describe the current status of Sec tRNAs in higher animals and further we examine: (i) the Sec tRNA population in Drosophila; (ii) transcription of the Sec tRNA in vivo (in Xenopus oocytes) and in vitro (in Xenopus oocyte extracts); (iii) the effect of selenium on the Sec tRNA population in various rat tissues following replenishment of extremely selenium deficient rats with this element; and (iv) the biosynthesis of the modified bases on Sec tRNA in Xenopus oocytes.
Asunto(s)
Biosíntesis de Proteínas , Proteínas , ARN de Transferencia Aminoácido-Específico/metabolismo , Animales , Secuencia de Bases , Mapeo Cromosómico , Drosophila/genética , Humanos , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/genética , Selenoproteínas , Serina-ARNt Ligasa/genética , XenopusRESUMEN
To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Proteínas , Aminoacil-ARN de Transferencia/metabolismo , Selenio/farmacología , Células Madre/enzimología , Animales , Southern Blotting , Cromatografía Líquida de Alta Presión , Clonación Molecular , Dosificación de Gen , Marcación de Gen , Vectores Genéticos , Glutatión Peroxidasa/biosíntesis , Heterocigoto , Ratones , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/genética , Recombinación Genética , Selenoproteínas , Células Madre/metabolismoRESUMEN
Selenoprotein P is an extracellular heparin-binding protein that has been implicated in protecting the liver against oxidant injury. Its location in liver, kidney, and brain was determined by conventional immunohistochemistry and confocal microscopy using a polyclonal antiserum. Selenoprotein P is associated with endothelial cells in the liver and is more abundant in central regions than in portal regions. It is also present in kidney glomeruli associated with capillary endothelial cells. Staining of selenoprotein P in the brain is also confined to vascular endothelial cells. The heparin-binding properties of selenoprotein P could be the basis for its binding to tissue. Its localization to the vicinity of endothelial cells is potentially relevant to its oxidant defense function.
Asunto(s)
Encéfalo/metabolismo , Endotelio/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Proteínas/metabolismo , Selenio , Animales , Encéfalo/citología , Endotelio/citología , Inmunohistoquímica , Riñón/citología , Hígado/citología , Masculino , Microscopía Confocal , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Selenoproteína P , SelenoproteínasRESUMEN
Selenoprotein P and glutathione peroxidase are selenoproteins that are synthesized by hepatocytes. The production of these selenoproteins by human and rat liver cell lines has been assessed at several levels of selenium supplementation and compared with one another. HepG2 and H4IIE cells were cultured in serum-free medium without selenium supplementation for 48 h; then sodium selenite was added to the medium to give final concentrations of 0, 1, 2.5, 5, or 10 ng selenium/ml medium. After 48 h, selenoprotein P concentration in the medium, cellular glutathione peroxidase activity, and the mRNA levels of the two selenoproteins were determined. Selenium deficiency caused a decrease in selenoprotein mRNA and protein levels. The extent of decrease depended on the cell line examined. In selenium-deprived HepG2 cells, selenoprotein P release decreased to 10% of the release by selenium-replete cells. Under the same conditions, cellular glutathione peroxidase activity decreased to 33%. H4IIE cells showed the opposite results with cellular glutathione peroxidase activity decreasing to 13% and selenoprotein P release decreasing to 40% of selenium-replete cells. The effect of dithiothreitol on secretion of selenoprotein P by H4IIE cells was examined. Selenoprotein P secretion was inhibited by dithiothreitol, suggesting that disulfide bond formation is necessary for secretion of the mature protein.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Hígado/metabolismo , Proteínas/metabolismo , Selenio/metabolismo , Animales , Cicloheximida/farmacología , Humanos , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Selenoproteína P , Selenoproteínas , Células Tumorales CultivadasRESUMEN
Several forms of selenoprotein P that share the same N-terminal sequence have been identified in rat plasma, but only one selenoprotein P mRNA has been characterized. The open reading frame of the mRNA contains 10 UGAs that presumably code for selenocysteine residues. Using heparin-Sepharose, we isolated two of the protein forms from immunoaffinity-purified selenoprotein P. One of the forms, Se-P45B, migrates at 45 kDa on SDS-polyacrylamide gel electrophoresis, and the other, Se-P57B, migrates at 57 kDa. These two forms were cleaved with cyanogen bromide, and both yielded 40-kDa fragments that were consistent with those fragments being an inter-methionine peptide near the N terminus of the predicted polypeptide. A 20-kDa fragment present in the cleavage products of Se-P57B was absent from the products of Se-P45B. This result suggested that Se-P45B lacks the C-terminal region of the predicted polypeptide. Carboxypeptidase P digestion of Se-P45B indicated that its C-terminal amino acid is Ser244, the amino acid immediately upstream from the predicted second selenocysteine. C-terminal analysis of Se-P57B indicated that its final residue is Asn366, the last amino acid predicted by the cDNA sequence. Amino acid composition analyses of the two forms were consistent with both arising from the same mRNA. Immunoaffinity-purified selenoprotein P was digested with proteases, and the resulting peptides were separated and sequenced. Only amino acid sequences predicted by the cDNA were found, and 80% of the predicted amino acid sequence was confirmed. These results are compatible with Se-P45B arising from termination of translation at the second in-frame UGA codon and all of the 10 in-frame UGA codons being read through to produce Se-P57B. These findings demonstrate that selenoprotein P isoforms of differing peptide lengths are present in plasma. They raise the possibility that the second UGA codon in selenoprotein P mRNA can have alternative functions: coding for the incorporation of selenocysteine or coding for termination of translation.
Asunto(s)
Proteínas/química , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Peso Molecular , Terminación de la Cadena Péptídica Traduccional , Fragmentos de Péptidos/química , Mapeo Peptídico , Ratas , Selenio , Selenoproteína P , SelenoproteínasRESUMEN
SDS-PAGE of immunoaffinity-purified rat seleno-protein P demonstrates a major band at 57 kDa and a less intense band at 45 kDa, suggesting the existence of more than one form of the protein. Separate experiments were carried out in which plasma from rats administered 75Se and immunoaffinity-purified 75Se-labeled selenoprotein P were applied to a heparin-Sepharose column at pH 7. In each experiment three peaks of 75Se-labeled protein were eluted during a continuous gradient from pH 7 to pH 8.5. The remaining 75Se-labeled material was eluted as a single peak by 1 M NaCl. Phosphorimaging of the SDS-PAGE gel of the peaks revealed that the first peak (1a) contained two bands (45 and 57 kDa) while the three remaining peaks each contained one band (peak 1b, 45 kDa and peaks 2 and 3, 57 kDa). N-terminal sequencing of the first eight amino acids of each band revealed that all five have the same N-terminal amino acid sequence and therefore are forms of selenoprotein P. Staining using a digoxigenin-based staining procedure revealed that all five forms contain carbohydrate. This demonstrates that plasma-derived selenoprotein P can be separated into five forms based on SDS-PAGE migration and heparin-Sepharose affinity and that at least two isoforms of selenoprotein P exist based on the presence of 45- and 57-kDa forms. The five forms have been given designations based on their order of elution from the heparin-Sepharose column and their M(r) on SDS-PAGE: selenoprotein P57A (peak 1a, 57 kDa), selenoprotein P45A (peak 1a, 45 kDa), selenoprotein P45B (peak 1b, 45 kDa), selenoprotein P57B (peak 2, 57 kDa), and selenoprotein P57C (peak 3, 57 kDa).
Asunto(s)
Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Digoxigenina , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Técnicas de Inmunoadsorción , Masculino , Datos de Secuencia Molecular , Peso Molecular , Proteínas/química , Ratas , Ratas Sprague-Dawley , Selenio/deficiencia , Radioisótopos de Selenio , Selenoproteína P , Selenoproteínas , Análisis de SecuenciaRESUMEN
Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M1, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.
Asunto(s)
Eritromicina/farmacología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Azitromicina/química , Azitromicina/farmacología , Farmacorresistencia Microbiana/genética , Eritromicina/química , Escherichia coli/genética , Mutación , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Relación Estructura-ActividadRESUMEN
The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.
Asunto(s)
Eritromicina/farmacología , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , ADN Bacteriano , Farmacorresistencia Microbiana/genética , Eritromicina/metabolismo , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.