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1.
Nucleic Acids Res ; 49(20): 11491-11511, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34648019

RESUMEN

The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection.


Asunto(s)
Codón Iniciador , Factor 3 de Iniciación Eucariótica/química , Proteínas Fúngicas/química , Iniciación de la Cadena Peptídica Traduccional , Ribosomas/ultraestructura , Microscopía por Crioelectrón , Factor 3 de Iniciación Eucariótica/metabolismo , Proteínas Fúngicas/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Kluyveromyces , Simulación de Dinámica Molecular , Unión Proteica , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Imagen Individual de Molécula
2.
Lett Appl Microbiol ; 76(1)2023 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-36688785

RESUMEN

The efficacy of Lactobacillus as an antibiotic substitute has been investigated as one of the potential strategies to prevent Salmonella infection in poultry. The purpose of this study was to explore the antibacterial activity of Lactobacillus fermentum 1.2133 (Lact. fermentum 1.2133) against Salmonella pullorum CVCC533 (Salm. pullorum CVCC533) and its effect on chickens infected with Salm. pullorum CVCC533. Results showed that Lact. fermentans 1.2133 has antibacterial activity against Salm. pullorum CVCC533 and the cell-free fermentation supernatant of Lact. fermentum 1.2133 had a bactericidal effect on the bacteria in the Salm. pullorum CVCC533 biofilm by significantly reducing the number of Salmonella and aerobic bacteria in the chicken duodenum, ileum, and cecum, including Escherichia shigella (P < 0.05), improved the species abundance of Lactobacilli (P < 0.05). The damage to the chicken intestine by Salm. pullorum CVCC533 was reduced as the expression of avian beta-defensin 2 (AvBD2) mRNA in chicken small intestine was increased (P < 0.05). The results showed that Lact. fermentum 1.2133 had the potential to be a probiotic for poultry due to its regulation of intestinal AvBD2 mRNA as well as its intestinal flora.


Asunto(s)
Limosilactobacillus fermentum , Enfermedades de las Aves de Corral , Probióticos , Salmonelosis Animal , Animales , Pollos/microbiología , Lactobacillus/fisiología , Salmonella , Aves de Corral , Antibacterianos
3.
Genes Dev ; 28(5): 502-20, 2014 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-24589778

RESUMEN

Eukaryotic initiator tRNA (tRNAi) contains several highly conserved unique sequence features, but their importance in accurate start codon selection was unknown. Here we show that conserved bases throughout tRNAi, from the anticodon stem to acceptor stem, play key roles in ensuring the fidelity of start codon recognition in yeast cells. Substituting the conserved G31:C39 base pair in the anticodon stem with different pairs reduces accuracy (the Sui(-) [suppressor of initiation codon] phenotype), whereas eliminating base pairing increases accuracy (the Ssu(-) [suppressor of Sui(-)] phenotype). The latter defect is fully suppressed by a Sui(-) substitution of T-loop residue A54. These genetic data are paralleled by opposing effects of Sui(-) and Ssu(-) substitutions on the stability of methionylated tRNAi (Met-tRNA(i)) binding (in the ternary complex [TC] with eIF2-GTP) to reconstituted preinitiation complexes (PICs). Disrupting the C3:G70 base pair in the acceptor stem produces a Sui(-) phenotype and also reduces the rate of TC binding to 40S subunits in vitro and in vivo. Both defects are suppressed by an Ssu(-) substitution in eIF1A that stabilizes the open/P(OUT) conformation of the PIC that exists prior to start codon recognition. Our data indicate that these signature sequences of tRNA(i) regulate accuracy by distinct mechanisms, promoting the open/P(OUT) conformation of the PIC (for C3:G70) or destabilizing the closed/P(IN) state (for G31:C39 and A54) that is critical for start codon recognition.


Asunto(s)
Codón Iniciador/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Iniciación de la Transcripción Genética , Emparejamiento Base , Secuencia Conservada , Mutación , Conformación Proteica , Estabilidad Proteica
4.
Nucleic Acids Res ; 47(21): 11326-11343, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31642471

RESUMEN

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.


Asunto(s)
Codón de Terminación/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Terminación de la Cadena Péptídica Traduccional , Proteínas Ribosómicas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Sitios de Unión/genética , Factor 3 de Iniciación Eucariótica/genética , Organismos Modificados Genéticamente , Terminación de la Cadena Péptídica Traduccional/genética , Unión Proteica , Biosíntesis de Proteínas/genética , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
5.
Proc Natl Acad Sci U S A ; 114(11): E2126-E2135, 2017 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-28223523

RESUMEN

The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNAiMet in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site ("POUT") to a closed, arrested conformation with TC tightly bound in the "PIN" state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions-R116D and R117D-also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG-anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC-mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.


Asunto(s)
Codón Iniciador , Complejos Multiproteicos/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Alelos , Sustitución de Aminoácidos , Factor 5 Eucariótico de Iniciación/química , Factor 5 Eucariótico de Iniciación/genética , Factor 5 Eucariótico de Iniciación/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Mutación , Fenotipo , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/química
6.
Nucleic Acids Res ; 42(15): 9623-40, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25114053

RESUMEN

eIF5 is the GTPase activating protein (GAP) for the eIF2 · GTP · Met-tRNAi (Met) ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2 · GDP · Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.


Asunto(s)
Codón Iniciador , Factor 5 Eucariótico de Iniciación/metabolismo , Guanosina Trifosfato/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Factor 1 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/genética , Factor 5 Eucariótico de Iniciación/química , Factor 5 Eucariótico de Iniciación/genética , Mutación , Fosfatos/metabolismo , Supresión Genética
8.
Genetics ; 220(1)2022 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-34791232

RESUMEN

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.


Asunto(s)
Saccharomyces cerevisiae
9.
Animals (Basel) ; 12(19)2022 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-36230269

RESUMEN

To understand the mechanism of lactic acid bacteria against Salmonella enteritidis infection; we examined how lactic acid bacteria regulated the intestinal microbiota to resist infection by pathogenic bacteria. The probiotic strain Lactobacillus reuteri S5 was used to construct an animal model of S. enteritidis infected broilers. A high-throughput sequencing technology was used to analyze the regulatory effects of L. reuteri S5 on the structure of the intestinal microbiota of broilers infected with S. enteritidis; and to examine the possible defense mechanism they used. Our results showed that the administration of L. reuteri S5 reduced colonization of S. enteritidis (p < 0.05), decreased intestinal permeability (p < 0.05), and reduced the bacterial displacement likely due by S. enteritidis colonization (p < 0.05), suggesting some enhancement of the intestinal barrier function. Furthermore, L. reuteri S5 increased the number of operational taxonomic units (OTUs) in the chicken cecal microflora and the relative abundance of Lactobacillaceae and decreased the relative abundance of Enterobacteriaceae. These results suggest that the lactic acid bacterium L. reuteri S5 protected the intestinal microbiota of chickens against S. enteritidis infection.

10.
3 Biotech ; 12(6): 126, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35573802

RESUMEN

Salmonella enteritidis is an important food-borne pathogen. The use of antibiotics is a serious threat to animal and human health, owing to the existence of resistant strains and drug residues. Lactic acid bacteria, as a new alternative to antibiotics, has attracted much attention. In this study, we investigated the antibacterial potential and underlying mechanism of Lactobacillus rhamnosus SQ511 against S. enteritidis ATCC13076. The results revealed that L. rhamnosus SQ511 significantly inhibited S. enteritidis ATCC13076 growth or even caused death. Laser confocal microscopic imaging revealed that the cell-free supernatant (CFS) of L. rhamnosus SQ511 elevated the reactive oxygen species level and bacterial membrane depolarization in S. enteritidis ATCC13076, leading to cell death. Furthermore, L. rhamnosus SQ511 CFS had severely deleterious effects on S. enteritidis ATCC13076, causing membrane destruction and the release of cellular materials. In addition, L. rhamnosus SQ511 CFS significantly reduced the expression of virulence, motility, adhesion, and invasion genes in S. enteritidis ATCC13076 (P < 0.05), and considerably inhibited motility and biofilm formation capacity (P < 0.05). Thus, antimicrobial compounds produced by L. rhamnosus SQ511 strongly inhibited S. enteritidis growth, mobility, biofilm formation, membrane disruption, and reactive oxygen species generation, and regulated virulence-related gene expressions, presenting promising applications as a probiotic agent.

11.
Mol Cell Biol ; 27(18): 6520-31, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17636014

RESUMEN

Nuclear cap binding complex (CBC) is recruited cotranscriptionally and stimulates spliceosome assembly on nascent mRNAs; however, its possible functions in regulating transcription elongation or termination were not well understood. We show that, while CBC appears to be dispensable for normal rates and processivity of elongation by RNA polymerase II (Pol II), it plays a direct role in preventing polyadenylation at weak termination sites. Similarly to Npl3p, with which it interacts, CBC suppresses the weak terminator of the gal10-Delta56 mutant allele by impeding recruitment of termination factors Pcf11p and Rna15p (subunits of cleavage factor IA [CF IA]) and does so without influencing Npl3p occupancy at the termination site. Importantly, deletion of CBC subunits or NPL3 also increases termination at a naturally occurring weak poly(A) site in the RNA14 coding sequences. We also show that CBC is most likely recruited directly to the cap of nascent transcripts rather than interacting first with transcriptional activators or the phosphorylated C-terminal domain of Pol II. Thus, our findings illuminate the mechanism of CBC recruitment and extend its function in Saccharomyces cerevisiae beyond mRNA splicing and degradation of aberrant nuclear mRNAs to include regulation of CF IA recruitment at poly(A) selection sites.


Asunto(s)
Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Modelos Biológicos , Unión Proteica , Saccharomyces cerevisiae/genética
12.
Mol Cell Biol ; 25(22): 9859-73, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16260602

RESUMEN

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/fisiología , Ribosomas/química , Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfato/química , Sitios de Unión , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al GTP/metabolismo , Genotipo , Proteínas Fluorescentes Verdes/metabolismo , Hidrólisis , Procesamiento de Imagen Asistido por Computador , Proteínas de Filamentos Intermediarios/metabolismo , Modelos Genéticos , Mutación , Fosfoproteínas/metabolismo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Ribosómico/química , ARN Ribosómico 18S/química , Proteínas Ribosómicas , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología
13.
Mol Cell Biol ; 25(8): 3063-75, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15798194

RESUMEN

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.


Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , eIF-2 Quinasa/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia Conservada , Análisis Mutacional de ADN , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/fisiología , Datos de Secuencia Molecular , Mutación/genética , Iniciación de la Cadena Peptídica Traduccional/fisiología , Fosforilación , Conformación Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Serina/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo
14.
Elife ; 62017 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-29206102

RESUMEN

The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor context. Ribosome profiling of NTT substitution R13P reveals heightened discrimination against unfavorable AUG context genome-wide. Both R13P and K16D substitutions destabilize the closed complex at UUG codons in reconstituted PICs. Thus, electrostatic interactions involving the eIF1A NTT stabilize the closed conformation and promote utilization of suboptimal start codons. We predict UM-associated mutations alter human gene expression by increasing discrimination against poor initiation sites.


Asunto(s)
Factor 1 Eucariótico de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Saccharomyces cerevisiae/metabolismo , Sustitución de Aminoácidos , Análisis Mutacional de ADN , Factor 1 Eucariótico de Iniciación/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , ARN Ribosómico 18S/metabolismo , ARN de Transferencia/metabolismo
15.
Nat Struct Mol Biol ; 18(11): 1227-34, 2011 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-22002225

RESUMEN

In contrast to prokaryotic elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the ribosomal A-site, eukaryotic initiation factor eIF2 binds methionyl initiator transfer RNA (Met-tRNA(i)(Met)) to the P-site of the 40S ribosomal subunit. The results of directed hydroxyl radical probing experiments to map binding of Saccharomyces cerevisiae eIF2 on the ribosome and on Met-tRNA(i)(Met) revealed that eIF2γ primarily contacts the acceptor stem of Met-tRNA(i)(Met) and identified a key binding interface between domain III of eIF2γ and 18S rRNA helix h44 on the 40S subunit. Whereas the analogous domain III of EF-Tu contacts the T stem of tRNAs, biochemical analyses demonstrated that eIF2γ domain III is important for ribosome, not Met-tRNA(i)(Met). Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new ribosome-binding function in eIF2γ.


Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Guanosina Trifosfato/metabolismo , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Cisteína/genética , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/genética , Guanosina Trifosfato/química , Radical Hidroxilo/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Ribosómico 18S/química , ARN Ribosómico 18S/metabolismo , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Mol Cell Biol ; 30(21): 5218-33, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20805354

RESUMEN

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(αP)]. While eIF2B contains five subunits, the ε/Gcd6 subunit is sufficient for GEF activity in vitro. The δ/Gcd2 and ß/Gcd7 subunits function with α/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(αP)-GDP, but the essential functions of δ/Gcd2 and ß/Gcd7 are not well understood. We show that the depletion of wild-type ß/Gcd7, three lethal ß/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in ß/Gcd7 and eIF2α all impair eIF2 binding to eIF2B without reducing ε/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, ß/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2α (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, ß/Gcd7 can overcome the toxicity of eIF2(αP) and rescue native eIF2B function when overexpressed with δ/Gcd2 or γ/Gcd1. In aggregate, these findings provide compelling evidence that ß/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (αP).


Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , Factor 2B Eucariótico de Iniciación/química , Factor 2B Eucariótico de Iniciación/genética , Genes Fúngicos , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Subunidades de Proteína , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
Genes Dev ; 22(16): 2242-55, 2008 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-18708582

RESUMEN

High-resolution structures of bacterial 70S ribosomes have provided atomic details about mRNA and tRNA binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (PICs). We identified residues in yeast 18S rRNA critical in vivo for recruiting methionyl tRNA(i)(Met) to 40S subunits during initiation by isolating mutations that derepress GCN4 mRNA translation. Several such Gcd(-) mutations alter the A928:U1389 base pair in helix 28 (h28) and allow PICs to scan through the start codons of upstream ORFs that normally repress GCN4 translation. The A928U substitution also impairs TC binding to PICs in a reconstituted system in vitro. Mutation of the bulge G926 in h28 and certain other residues corresponding to direct contacts with the P-site codon or tRNA in bacterial 70S complexes confer Gcd(-) phenotypes that (like A928 substitutions) are suppressed by overexpressing tRNA(i)(Met). Hence, the nonconserved 928:1389 base pair in h28, plus conserved 18S rRNA residues corresponding to P-site contacts in bacterial ribosomes, are critical for efficient Met-tRNA(i)(Met) binding and AUG selection in eukaryotes.


Asunto(s)
Codón Iniciador/genética , Iniciación de la Cadena Peptídica Traduccional , ARN de Hongos/genética , ARN Ribosómico 18S/genética , ARN de Transferencia de Metionina/genética , Saccharomyces cerevisiae/genética , Sustitución de Aminoácidos , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Mutación/genética , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
J Biol Chem ; 281(11): 7452-7, 2006 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-16421098

RESUMEN

The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2alpha and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Chaperoninas/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Acetilcisteína/metabolismo , Secuencia de Aminoácidos , Animales , División Celular , Línea Celular , Chaperoninas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/química , Evolución Molecular , VIH/metabolismo , Células HeLa , Humanos , Immunoblotting , Interferones/metabolismo , Intrones , Datos de Secuencia Molecular , Familia de Multigenes , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasas/química , Homología de Secuencia de Aminoácido , Virus de la Inmunodeficiencia de los Simios , Xenopus , Xenopus laevis , Factor 5A Eucariótico de Iniciación de Traducción
19.
Genes Dev ; 16(10): 1271-80, 2002 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-12023305

RESUMEN

The protein kinase GCN2 is activated in amino acid-starved cells on binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related domain. We isolated two point mutations in the protein kinase (PK) domain, R794G and F842L, that permit strong kinase activity in the absence of tRNA binding. These mutations also bypass the requirement for ribosome binding, dimerization, and association with the GCN1/GCN20 regulatory complex, suggesting that all of these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the N terminus of the HisRS segment. As Arg 794 and Phe 842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Autophosphorylation of the activation loop was stimulated by R794G and F842L, and the autophosphorylation sites remained critical for GCN2 function in the presence of these mutations. Our results imply a two-step activation mechanism involving distinct conformational changes in the PK domain.


Asunto(s)
Mutación/genética , Proteínas Quinasas/metabolismo , ARN de Transferencia/metabolismo , Sitios de Unión , Dimerización , Activación Enzimática , Regulación de la Expresión Génica , Histidina-ARNt Ligasa/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Quinasas/genética , Estructura Terciaria de Proteína
20.
J Biol Chem ; 279(40): 42157-68, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15277527

RESUMEN

RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors. RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes. Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size. There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal. A mutation in the second ATP-binding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts. These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation. RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells. Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Fúngicas/fisiología , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas de Ciclo Celular/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Sustancias Macromoleculares , Unión Proteica , Ribosomas , Proteínas de Saccharomyces cerevisiae/metabolismo
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