RESUMEN
The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542-883). Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus. This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different. We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth. Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1-eIF4E complex, but not by eIF4E alone. Analysis of mutant eIF4G(542-883) proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G. We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G(542-883) proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A. Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Factor 4A Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Mamíferos , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/química , Unión Proteica , Biosíntesis de Proteínas , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
Eukaryotic initiation factor (eIF) 4A is an essential protein that, in conjunction with eIF4B, catalyzes the ATP-dependent melting of RNA secondary structure in the 5'-untranslated region of mRNA during translation initiation. In higher eukaryotes, eIF4A is assumed to be recruited to the mRNA through its interaction with eIF4G. However, the failure to detect this interaction in yeast brought into question the generality of this model. The work presented here demonstrates that yeast eIF4G interacts with eIF4A both in vivo and in vitro. The eIF4A-binding site was mapped to amino acids 542-883 of yeast eIF4G1. Expression in yeast cells of the eIF4G1 domain that binds eIF4A results in cell growth inhibition, and addition of this domain to an eIF4A-dependent in vitro system inhibits translation in a dose-dependent manner. Both in vitro translation and cell growth can be specifically restored by increasing the eIF4A concentration. These data demonstrate that yeast eIF4A and eIF4G interact and suggest that this interaction is required for translation and cell growth.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor 4A Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Factores de Iniciación de Péptidos/genética , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Factor 3 de Iniciación Eucariótica , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis , Factores de Iniciación de Péptidos/química , Fenotipo , Factor 3 Procariótico de Iniciación , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
BACKGROUND: Translation initiation factor 4A (elF4A) is the prototype of the DEAD-box family of proteins. DEAD-box proteins are involved in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. Energy from ATP hydrolysis is used to perform RNA unwinding during initiation of mRNA translation. The presence of elF4A is required for the 43S preinitiation complex to bind to and scan the mRNA. RESULTS: We present here the crystal structure of the nucleotide-binding domain of elF4A at 2.0 A and the structures with bound adenosinediphosphate and adenosinetriphosphate at 2.2 A and 2.4 A resolution, respectively. The structure of the apo form of the enzyme has been determined by multiple isomorphous replacement. The ATPase domain contains a central seven-stranded beta sheet flanked by nine alpha helices. Despite low sequence homology to the NTPase domains of RNA and DNA helicases, the three-dimensional fold of elF4A is nearly identical to the DNA helicase PcrA of Bacillus stearothermophilus and to the RNA helicase NS3 of hepatitis C virus. CONCLUSIONS: We have determined the crystal structure of the N-terminal domain of the elF4A from yeast as the first structure of a member of the DEAD-box protein family. The complex of the protein with bound ADP and ATP offers insight into the mechanism of ATP hydrolysis and the transfer of energy to unwind RNA. The identical fold of the ATPase domain of the DNA helicase PcrA of B. stearothermophilus and the RNA helicase of hepatitis C virus suggests a common fold for all ATPase domains of DExx- and DEAD-box proteins.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas , Factores de Iniciación de Péptidos/química , Saccharomyces cerevisiae/química , Adenosina Difosfato/química , Adenosina Trifosfato/química , Cristalografía por Rayos X , Proteínas Fúngicas/química , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Subtilisinas/química , Proteínas no Estructurales Virales/químicaRESUMEN
The essential cap-binding protein (eIF4E) of Saccharomyces cerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 degrees C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5'-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5'-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5'-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.
Asunto(s)
Ciclinas/fisiología , Proteínas Fúngicas/fisiología , Fase G1 , Mutación , Factores de Iniciación de Péptidos/fisiología , Fase S , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Regiones no Traducidas 5'/genética , Ciclinas/genética , Factor 4E Eucariótico de Iniciación , Citometría de Flujo , Proteínas Fúngicas/genética , Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Genes Reporteros , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Biosíntesis de Proteínas , Proteínas Quinasas/genética , ARN Mensajero/genética , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Alelos , Secuencia de Bases , Cartilla de ADN , Factor 2B Eucariótico de Iniciación , Factores de Intercambio de Guanina Nucleótido , Mutación , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The Saccharomyces cerevisiae TIF3 gene encodes a 436-amino acid (aa) protein that is the yeast homologue of mammalian translation Initiation factor eIF4B. Tif3p can be divided into three parts, the N-terminal region with an RNA recognition motif (RRM) (aa 1-182), followed in the middle part by a sevenfold repeat of 26 amino acids rich in basic and acidic residues (as 183-350), and a C-terminal region without homology to any known sequence (aa 351-436). We have analyzed several Tif3 proteins with deletions at their N and C termini for their ability (1) to complement a tif3delta strain in vivo, (2) to stimulate Tif3-dependent translation extracts, (3) to bind to single-stranded RNA, and (4) to catalyze RNA strand-exchange in vitro. Here we report that yeast Tif3/eIF4B contains at least two RNA binding domains able to bind to single-stranded RNA. One is located in the N-terminal region of the protein carrying the RRM, the other in the C-terminal two-thirds region of Tif3p. The RRM-containing domain and three of the seven repeat motifs are essential for RNA strand-exchange activity of Tif3p and translation in vitro and for complementation of a tif3delta strain, suggesting an important role for RNA strand-exchange activity in translation.
Asunto(s)
Proteínas Fúngicas/química , Iniciación de la Cadena Peptídica Traduccional/genética , Factores de Iniciación de Péptidos , ARN Nucleotidiltransferasas/química , ARN Mensajero/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sistema Libre de Células , Factores Eucarióticos de Iniciación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Unión Proteica , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión , Eliminación de SecuenciaRESUMEN
Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43 S preinitiation complex by dissociating 40 and 60 S ribosomal subunits, stabilizing the ternary complex, and promoting mRNA binding to 40 S ribosomal subunits. The product of the Saccharomyces cerevisiae RPG1 gene has been described as encoding a protein required for passage through the G1 phase of the cell cycle and exhibiting significant sequence similarity to the largest subunit of human eIF3. Here we show that under nondenaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p. An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc tag of a tagged version of Prt1p co-immunoprecipitates Rpg1p, demonstrating that both proteins are present in the same complex. A cell-free translation system derived from the temperature-sensitive rpg1-1 mutant strain becomes inactivated by incubation at 37 degreesC, and its activity can be restored by the addition of the Rpg1-containing protein complex. Finally, the rpg1-1 temperature-sensitive mutant strain shows a dramatic reduction of the polysome/monosome ratio upon shift to the restrictive temperature. These data show that Rpg1p is an authentic eIF3 subunit and plays an important role in the initiation step of translation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Factor 3 de Iniciación Eucariótica , Proteínas Fúngicas/genética , Genes Fúngicos , Pruebas de Precipitina , Saccharomyces cerevisiae/citologíaRESUMEN
Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5' cap-dependent pathway. It interacts with eIF4E (the mRNA 5' cap-binding protein) and serves as an anchor for the assembly of further initiation factors. With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable. We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Polienos/farmacología , Biosíntesis de Proteínas , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Ciclo Celular/efectos de los fármacos , Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Genotipo , Cinética , Factores de Iniciación de Péptidos/efectos de los fármacos , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus , Factores de TiempoRESUMEN
Translation initiation factor-dependent extracts are prepared from Saccharomyces cerevisiae strains that have no or reduced activity of a translation initiation factor. Elimination of factor activity can be achieved by deletion of the gene encoding the factor if it is not essential for the survival of the strain. If the gene is essential it is placed under the control of the regulatable GAL1 promoter and its expression is shut off in vivo. Alternatively, a temperature-sensitive mutation can be introduced into the gene and the activity of the gene product eliminated in vitro by preincubation of the extract at the nonpermissive temperature. Factor-dependent extracts can be complemented in vitro with purified initiation factor preparations isolated from S. cerevisiae or from Escherichia coli cells expressing them from plasmid-encoded constructs. To simplify the purification of the factors they may be expressed as fusion proteins with N- or C-terminal tags. Initiation factor-dependent extracts can be used to study initiation factor structure-function relationships and initiation factor requirements for specific mRNA translation.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Mutación , Factores de Iniciación de Péptidos/química , Biosíntesis de Proteínas , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , Temperatura , TransfecciónRESUMEN
In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein. We demonstrate here that p20 is a repressor of cap-dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F-complex.
Asunto(s)
Proteínas Fúngicas/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , División Celular , Electroforesis en Gel de Poliacrilamida , Factor 4E Eucariótico de Iniciación , Factor 4F Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
A 45-year-old Greek patient was found to have a moderate iron overload (ferritin 1213 micrograms/l, serum iron 21.5 mumol/l, transferrin saturation 40%). He underwent 12 phlebotomies of 450 cc over an 8-year period and ferritin was normalized (267 micrograms/l) after the seventh. Study of the HLA-H gene in leukocyte DNA showed that the patient is homozygous for the His63Asp mutation while no modification was found at position 282. This case is compared with that of a 50-year-old Swiss male presenting a severe iron overload (ferritin 7660 micrograms/l, serum iron 36.5 mumol/l, transferrin saturation 97%). Although this patient has undergone 77 phlebotomies (450 cc each time) over a 2-year period, he continues to have a high ferritin level (2200 micrograms/l). HLA-H gene analysis showed the absence of codon 63 mutation and the presence of Cys282Tyr mutation in the homozygous state. The study of these two cases indicates that penetrance of the His63Asp mutation in the homozygous state is very low as compared to Cys282Tyr and results in moderate iron accumulation, probably without organ damage. This genotype must be looked for whenever moderate iron overload is present.
Asunto(s)
Análisis Mutacional de ADN , Antígenos HLA-A/genética , Hemocromatosis/genética , Homocigoto , Ferritinas/metabolismo , Hemocromatosis/sangre , Humanos , Hierro/sangre , Masculino , Persona de Mediana Edad , FlebotomíaRESUMEN
The Saccharomyces cerevisiae TIF3 gene encodes the yeast homologue of mammalian translation initiation factor eIF-4B. We have added six histidine residues to the C-terminus of Tif3 protein (Tif3-His6p) and purified the tagged protein by affinity chromatography. Tif3-His6p stimulates translation and mRNA binding to ribosomes in a Tif3-dependent in vitro system. Furthermore, it binds to single-stranded RNA and catalyses the annealing of partially complementary RNA strands in vitro. In parallel experiments, RNA annealing activity could also be demonstrated for mammalian eIF-4B. A role for Tif3/eIF-4B and RNA annealing activity in the scanning process is proposed.
Asunto(s)
Factores Eucarióticos de Iniciación , Proteínas Fúngicas/metabolismo , Iniciación de la Cadena Peptídica Traduccional/genética , Factores de Iniciación de Péptidos/metabolismo , ARN Nucleotidiltransferasas/metabolismo , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animales , Secuencia de Bases , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Histidina/genética , Modelos Genéticos , Datos de Secuencia Molecular , Renaturación de Ácido Nucleico , Factores de Iniciación de Péptidos/genética , ARN Nucleotidiltransferasas/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ribosomas/metabolismo , Eliminación de SecuenciaRESUMEN
Translation initiation factor Prt1 was purified from a ribosomal salt wash fraction of Saccharomyces cerevisiae cells by ammonium sulfate precipitation, DEAE chromatography, phosphocellulose chromatography, sucrose density gradient centrifugation, and non-denaturing polyacrylamide gel electrophoresis. Prt1 protein cofractionates with four other polypeptides during all steps of purification suggesting that it is part of a protein complex containing polypeptide subunits with apparent molecular masses of 130, 80, 75 (Prt1), 40, and 32 kDa. Deletion of the first AUG codon in the published sequence of the PRT1 gene results in the synthesis of functional Prt1 protein indicating that the actual molecular mass of the Prt1 subunit is 82.7 kDa. This is in agreement with results from primer extension experiments reported earlier by Keierleber et al. (Keierleber, C., Wittekind, M., Qin, S., and McLaughlin, C. S. (1986) Mol. Cell. Biol. 6, 4419-4424). The Prt1-containing protein complex is an active translation factor as shown by its ability to restore translation in a cell-free system derived from a temperature-sensitive prt1 mutant strain in which endogenous Prt1 activity is inactivated by heating the extract to 37 degrees C. The question of whether the Prt1-containing protein complex represents the yeast homologue of mammalian translation initiation factor eIF-3 is discussed.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Proteínas Fúngicas/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Secuencia de Bases , Cromatografía por Intercambio Iónico , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genéticaRESUMEN
Characterization of cDNAs encoding eukaryotic translation initiation factor 4A (eIF-4A) indicates the expression of a minimum of ten related genes in tobacco leaf cells. The ten groups fall into two gene families, NeIF-4A2 and NeIF-4A3. The majority of the cDNAs exhibit significant sequence similarity to the NeIF-4A2 family at both the DNA and deduced amino acid levels. Northern analysis using specific probes indicates variable expression of four family members in various tobacco organs. Western analysis, using an anti-tobacco eIF-4A polyclonal antibody, reveals a complex pattern of immunologically related polypeptides of approximately 46 kDa. Subcellular fractionation suggests that at least one eIF-4A-related polypeptide is located in the chloroplast where it is ribosome-associated.
Asunto(s)
Genes de Plantas/genética , Familia de Multigenes/genética , Nicotiana/genética , Factores de Iniciación de Péptidos/genética , Plantas Tóxicas , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Compartimento Celular , ADN Complementario/genética , Factor 4A Eucariótico de Iniciación , Dosificación de Gen , Biblioteca de Genes , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Factores de Iniciación de Péptidos/inmunología , Factores de Iniciación de Péptidos/aislamiento & purificación , Semillas/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Triticum/químicaRESUMEN
This review summarizes recent progress in the study of initiation of protein synthesis in the yeast Saccharomyces cerevisiae. Biochemical and genetic approaches provide new insight into the recognition of the 5'-end of mRNA by initiation factors and 40S ribosomes, unwinding of mRNA secondary structures in the untranslated region and proper recognition of the AUG start codon. Experiments with initiation factor-dependent cell-free systems have facilitated studies of factor functions and factor requirements for translation of different mRNAs. The analysis of mutations which suppress the inhibitory effect on translation of RNA secondary structure in the 5'-untranslated region of yeast mRNAs has led to the identification of gene products which may be involved in both transcription and translation.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Factores de Iniciación de Péptidos/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Eucariotas/metabolismoRESUMEN
Translational control of gene expression is an important regulatory mechanism in cellular physiology. In eukaryotes, ribosomes can initiate translation by two different mechanisms: a majority of mRNAs undergo cap-dependent initiation at their extreme 5'-ends, but initiation can occur internally in some mRNAs. A number of important cellular responses, such as entry into a proliferative state and adaptation to changing nutrient levels, are mediated by changes in the mechanism of translation initiation of specific mRNAs. This article discusses new insights into control of gene expression gained through studies of regulation of eukaryotic translational initiation.
Asunto(s)
Proteínas de Unión al ADN , Regulación de la Expresión Génica/fisiología , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , División Celular , Proteínas Fúngicas/metabolismo , Hierro/metabolismo , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae , Factores de Transcripción/metabolismoRESUMEN
The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced. The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF-4B. The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold-sensitive phenotype. In vitro translation of total yeast RNA in an extract from a TIF3 gene-disrupted strain is reduced compared with a wild-type extract. The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild-type extract or mammalian eIF-4B, but not by addition of mutant extract. In vivo translation of beta-galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5' leader region in a TIF3 gene-disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure. This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo.
Asunto(s)
Factores Eucarióticos de Iniciación , Proteínas Fúngicas/genética , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , ADN de Hongos , Factor 3 de Iniciación Eucariótica , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Cinética , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/metabolismo , ARN Helicasas , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia de Consenso , Factor 4F Eucariótico de Iniciación , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas de Unión a Caperuzas de ARN , Ribosomas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.