RESUMEN

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.

Asunto(s)

Factor 1 Eucariótico de Iniciación , Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/fisiología , Ribosomas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Sitios de Unión , Factor 5 Eucariótico de Iniciación , Evolución Molecular , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/genética , Estructura Terciaria de Proteína , ARN de Transferencia de Metionina/metabolismo , Alineación de Secuencia

RESUMEN

The protein kinase PKR (dsRNA-dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2alpha to downregulate protein synthesis in virus-infected cells. Two double-stranded RNA binding domains (dsRBDs) in the N-terminal half of PKR are thought to bind the activator double-stranded RNA, mediate dimerization of the protein and target PKR to the ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to heterologous dimerization domains. Whereas the isolated PKR kinase domain (KD) was non-functional in vivo, expression of a glutathione S-transferase-KD fusion, or co-expression of KD fusions containing the heterodimerization domains of the Xlim-1 and Ldb1 proteins, restored PKR activity in yeast cells. Finally, coumermycin-mediated dimerization of a GyrB-KD fusion protein increased eIF2alpha phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of dimerization for PKR activity in vivo, and suggest that a primary function of double-stranded RNA binding to the dsRBDs of native PKR is to promote dimerization and activation of the kinase domain.

Asunto(s)

ARN Bicatenario/metabolismo , eIF-2 Quinasa/metabolismo , Células 3T3 , Animales , Sitios de Unión , Dimerización , Activación Enzimática , Ratones , Saccharomyces cerevisiae/enzimología , eIF-2 Quinasa/química

RESUMEN

The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.

Asunto(s)

Proteínas Fúngicas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Poli A/genética , Biosíntesis de Proteínas , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , ARN Helicasas DEAD-box , Electroporación , Factor 5 Eucariótico de Iniciación , Eliminación de Gen , Genes Reporteros , Cinética , Modelos Biológicos , Mutación , Proteínas de Unión a Poli(A) , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supresión Genética , Factores de Tiempo

RESUMEN

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha subunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the alpha subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2alpha (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alphaP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(alphaP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-GTP exchange.

Asunto(s)

Factor 2B Eucariótico de Iniciación/química , Factor 2B Eucariótico de Iniciación/metabolismo , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Sitios de Unión , Catálisis , Genotipo , Glutatión Transferasa/metabolismo , Modelos Biológicos , Mutación , Níquel/metabolismo , Fosforilación , Plásmidos/metabolismo , Factor 2 Procariótico de Iniciación , Unión Proteica , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , ARN de Transferencia de Metionina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) is a conserved mechanism regulating protein synthesis in response to various stresses. A screening for negative factors in yeast salt stress tolerance has led to the identification of Gcn2p, the single yeast eIF2alpha kinase that is activated by amino acid starvation in the general amino acid control response. Mutation of other components of this regulatory circuit such as GCN1 and GCN3 also resulted in improved NaCl tolerance. The gcn2 phenotype was not accompanied by changes in sodium or potassium homeostasis. NaCl induced a Gcn2p-dependent phosphorylation of eIF2alpha and translational activation of Gcn4p, the transcription factor that mediates the general amino acid control response. Mutations that activate Gcn4p function, such as gcd7-201, cpc2, and deletion of the translational regulatory region of the GCN4 gene, also cause salt sensitivity. It can be postulated that sodium activation of the Gcn2p pathway has toxic effects on growth under NaCl stress and that this novel mechanism of sodium toxicity may be of general significance in eukaryotes.

Asunto(s)

Proteínas de Unión al ADN , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efectos de los fármacos , Sodio/toxicidad , Aminoácidos/farmacología , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación , Proteínas Fúngicas/fisiología , Nucleotidasas/genética , Factores de Elongación de Péptidos , Fosforilación , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo

RESUMEN

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.

Asunto(s)

ARN Bicatenario/metabolismo , eIF-2 Quinasa/metabolismo , Secuencia de Aminoácidos , Dimerización , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Plásmidos , Poli I-C/metabolismo , Treonina/metabolismo , Levaduras/enzimología , eIF-2 Quinasa/genética

RESUMEN

We have previously reported a physical association between STAT1 and the protein kinase double-stranded RNA-activated protein kinase (PKR). PKR inhibited STAT1 function in a manner independent of PKR kinase activity. In this report, we have further characterized the properties of both molecules by mapping the sites of their interaction. A STAT1 mutant unable to interact with PKR displays enhanced interferon gamma (IFN-gamma)-induced transactivation capacity compared with STAT1. This effect appears to be mediated by the higher capacity of STAT1 mutant to heterodimerize with STAT3. Furthermore, expression of STAT1 mutant in STAT1(-/-) cells enhances both the antiviral and antiproliferative effects of IFNs as opposed to STAT1. We also provide evidence that STAT1 functions as an inhibitor of PKR in vitro and in vivo. That is, phosphorylation of eIF-2alpha is enhanced in STAT1(-/-) than STAT1(+/+) cells in vivo, and this correlates with higher activation capacity of PKR in STAT1(-/-) cells. Genetic experiments in yeast demonstrate the inhibition of PKR activation and eIF-2alpha phosphorylation by STAT1 but not by STAT1 mutant. These data substantiate our previous findings on the inhibitory effects of PKR on STAT1 and implicate STAT1 in translational control through the modulation of PKR activation and eIF-2alpha phosphorylation.

Asunto(s)

Antivirales/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación , Transactivadores/genética , Transactivadores/metabolismo , eIF-2 Quinasa/metabolismo , Aminoácidos/química , Sitios de Unión , División Celular/efectos de los fármacos , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/química , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Immunoblotting , Interferón gamma/metabolismo , Mutagénesis , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Biosíntesis de Proteínas , Factor de Transcripción STAT1 , Factores de Tiempo , Transactivadores/química , Transcripción Genética , Activación Transcripcional , Transfección

Asunto(s)

Factor 2 Eucariótico de Iniciación/genética , Factores Eucarióticos de Iniciación/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Biosíntesis de Proteínas/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/metabolismo , Guanosina Trifosfato/metabolismo , Methanobacterium/genética , Modelos Moleculares , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/metabolismo , Conformación Proteica , Ribosomas/genética , Ribosomas/metabolismo

RESUMEN

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.

Asunto(s)

Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Factores de Iniciación de Péptidos/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Escherichia coli/metabolismo , Factor 5 Eucariótico de Iniciación , Guanina/metabolismo , Methanococcus/química , Modelos Moleculares , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

The interferon-induced protein kinase PKR is activated upon binding double-stranded RNA and phosphorylates the translation initiation factor eIF2alpha on Ser-51 to inhibit protein synthesis in virally infected cells. Swinepox virus C8L and vaccinia virus K3L gene products structurally resemble the amino-terminal third of eIF2alpha. We demonstrate that the C8L protein, like the K3L protein, can reverse the toxic effects caused by high level expression of human PKR in yeast cells. In addition, expression of either the K3L or C8L gene product was found to reverse the inhibition of reporter gene translation caused by PKR expression in mammalian cells. The inhibitory function of the K3L and C8L gene products in these assays was found to be critically dependent on residues near the carboxyl-termini of the proteins including a sequence motif shared among eIF2alpha and the C8L and K3L gene products. Thus, despite significant sequence differences both the C8L and K3L proteins function as pseudosubstrate inhibitors of PKR.

Asunto(s)

Suipoxvirus/genética , Proteínas Virales/genética , eIF-2 Quinasa/antagonistas & inhibidores , Células 3T3 , Secuencia de Aminoácidos , Animales , Inhibidores Enzimáticos/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Fosforilación , Biosíntesis de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Suipoxvirus/metabolismo , Transfección , Proteínas Virales/química , Proteínas Virales/metabolismo , eIF-2 Quinasa/metabolismo

RESUMEN

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

Asunto(s)

Proteínas Bacterianas/fisiología , Células Eucariotas/metabolismo , Factor 1 Eucariótico de Iniciación/fisiología , Factor 2 Eucariótico de Iniciación/fisiología , Iniciación de la Cadena Peptídica Traduccional/fisiología , Factores de Iniciación de Péptidos/fisiología , Células Procariotas/metabolismo , Escherichia coli/genética , Factor 5 Eucariótico de Iniciación , Sustancias Macromoleculares , Imitación Molecular , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/genética , Fenotipo , Factor 1 Procariótico de Iniciación , Unión Proteica , Estructura Terciaria de Proteína , ARN de Transferencia/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Ribosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , Relación Estructura-Actividad , Técnicas del Sistema de Dos Híbridos

RESUMEN

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Asunto(s)

Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/metabolismo , Puromicina/análogos & derivados , Ribosomas/metabolismo , Secuencia de Aminoácidos , Catálisis , Codón Iniciador , Factor 1 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica , Factor 5 Eucariótico de Iniciación , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Hidrólisis , Datos de Secuencia Molecular , Puromicina/biosíntesis , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

The family of eukaryotic initiation factor 2alpha (eIF2alpha) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the alpha subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2alpha kinases was examined by substituting Ser-51 in eIF2alpha with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2alpha-S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2alpha followed by detection using anti-eIF2alpha and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2alpha-S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2alpha kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways.

Asunto(s)

Factor 2 Eucariótico de Iniciación/metabolismo , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , eIF-2 Quinasa/metabolismo , Alelos , Línea Celular , Factor 2 Eucariótico de Iniciación/química , Humanos , Focalización Isoeléctrica , Fosforilación , Biosíntesis de Proteínas , Especificidad por Sustrato

RESUMEN

Initiation of protein synthesis requires both an mRNA and the initiator methionyl (Met)-tRNA to be bound to the ribosome. Most mRNAs are recruited to the ribosome through recognition of the 5' m7G cap by a group of proteins referred to as the cap-binding complex or eIF4F. Evidence is accumulating that eIF4G, the largest subunit of the cap-binding complex, serves as a central adapter by binding to various translation factors and regulators. Other translation factors also have modular structures that facilitate multiple protein-protein interactions, which suggests that adapter functions are common among the translation initiation factors. By linking different regulatory domains to a conserved eIF2-kinase domain, cells adapt to stress and changing growth conditions by altering the translational capacity through phosphorylation of eIF2, which mediates the binding of the initiator Met-tRNA to the ribosome.

Asunto(s)

Biosíntesis de Proteínas , Factor 4G Eucariótico de Iniciación , Estrés Oxidativo , Factores de Iniciación de Péptidos/fisiología , eIF-2 Quinasa/metabolismo

RESUMEN

Binding of initiator methionyl-tRNA to ribosomes is catalyzed in prokaryotes by initiation factor (IF) IF2 and in eukaryotes by eIF2. The discovery of both IF2 and eIF2 homologs in yeast and archaea suggested that these microbes possess an evolutionarily intermediate protein synthesis apparatus. We describe the identification of a human IF2 homolog, and we demonstrate by using in vivo and in vitro assays that human IF2 functions as a translation factor. In addition, we show that archaea IF2 can substitute for its yeast homolog both in vivo and in vitro. We propose a universally conserved function for IF2 in facilitating the proper binding of initiator methionyl-tRNA to the ribosomal P site.

Asunto(s)

Archaea/genética , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/genética , Factores de Iniciación de Péptidos , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia Conservada , Humanos , Cinética , Datos de Secuencia Molecular , Filogenia , Factor 2 Procariótico de Iniciación , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética

RESUMEN

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel, Spermophilus tridecemlineatus. PS, in vivo, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37 degreesC, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 +/- 0.08 pmol/mg protein per min; hibernator, 0.16 +/- 0.05 pmol/mg protein per min, P < 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2alpha are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 +/- 0.7 min; hibernator, 7.1 +/- 1.4 min, P < 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate "trickle" blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.

Asunto(s)

Encéfalo/metabolismo , Hibernación/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Extensión de la Cadena Peptídica de Translación , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Ribosomas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Autorradiografía/métodos , Radioisótopos de Carbono , Factor 2 Eucariótico de Iniciación/metabolismo , Leucina/metabolismo , Proteínas del Tejido Nervioso/aislamiento & purificación , Factor 2 de Elongación Peptídica , Factores de Elongación de Péptidos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Sciuridae , Sensibilidad y Especificidad , Transcripción Genética

RESUMEN

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Proteínas de Unión al ARN/genética , Proteínas Virales/genética , eIF-2 Quinasa/genética , División Celular/genética , Dimerización , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Mutación/genética , Fosforilación , Unión Proteica/genética , Proteínas Recombinantes de Fusión/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética

Asunto(s)

Saccharomyces cerevisiae/enzimología , eIF-2 Quinasa/análisis , Animales , Humanos

RESUMEN

Delivery of the initiator methionine transfer RNA (Met-tRNAiMet) to the ribosome is a key step in the initiation of protein synthesis. Previous results have indicated that this step is catalyzed by the structurally dissimilar translation factors in prokaryotes and eukaryotes-initiation factor 2 (IF2) and eukaryotic initiation factor 2 (eIF2), respectively. A bacterial IF2 homolog has been identified in both eukaryotes and archaea. By using a combination of molecular genetic and biochemical studies, the Saccharomyces cerevisiae IF2 homolog is shown to function in general translation initiation by promoting Met-tRNAiMet binding to ribosomes. Thus, the mechanism of protein synthesis in eukaryotes and prokaryotes is more similar than was previously realized.

Asunto(s)

Proteínas de Unión al ADN , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia de Metionina/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Codón Iniciador , Citoplasma/química , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/farmacología , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/análisis , Factores de Iniciación de Péptidos/genética , Factor 2 Procariótico de Iniciación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo

RESUMEN

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2alpha kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2alpha phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2alpha kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

Asunto(s)

Baculoviridae/enzimología , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Virales/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Línea Celular , Eliminación de Gen , Vectores Genéticos , Factores de Intercambio de Guanina Nucleótido , Humanos , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Spodoptera , Transfección , Proteínas Virales/genética , Replicación Viral , eIF-2 Quinasa/biosíntesis