Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
J Biol Chem ; 298(7): 102133, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35700825

RESUMO

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.


Assuntos
Nucleotídeos , Biossíntese de Proteínas , Animais , Códon de Terminação/genética , Códon de Terminação/metabolismo , Eucariotos/metabolismo , Humanos , Mamíferos/metabolismo , Nucleotídeos/genética , Nucleotídeos/metabolismo , Elongação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
2.
Nucleic Acids Res ; 49(19): 11181-11196, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34591963

RESUMO

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions - binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.


Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Proteína I de Ligação a Poli(A)/genética , Ribossomos/genética , Animais , Sistema Livre de Células , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Fatores de Terminação de Peptídeos/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reticulócitos/química , Reticulócitos/metabolismo , Ribossomos/metabolismo , Transdução de Sinais
3.
Mol Cell ; 53(4): 645-54, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24486019

RESUMO

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.


Assuntos
Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Oxigenases de Função Mista/química , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/química , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Catálise , Linhagem Celular Tumoral , Códon de Terminação , Células HeLa , Humanos , Hidrólise , Hidroxilação , Histona Desmetilases com o Domínio Jumonji , Modelos Moleculares , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
4.
RNA Biol ; 18(sup2): 804-817, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34793288

RESUMO

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.


Assuntos
Biossíntese de Proteínas , SARS-CoV-2 , Proteínas não Estruturais Virais/fisiologia , Animais , Sistema Livre de Células , Códon de Terminação/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Mutação , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Peptídeos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/metabolismo , Coelhos , Ribossomos/metabolismo
5.
J Biol Chem ; 294(21): 8630-8639, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30992367

RESUMO

Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination in vitro However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.


Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Humanos , Fatores de Iniciação de Peptídeos/química , Fatores de Terminação de Peptídeos/química , Poli A/química , Poli A/metabolismo , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas Repressoras/química
6.
Methods ; 162-163: 54-59, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31201933

RESUMO

Classical toeprinting is generally used to determine the position of ribosomes on mRNA; however, it has several disadvantages. We describe a fluorescent toeprinting assay that enables easier identification of ribosomal complexes bound to mRNA in vitro. The procedure involves the use of stable and safe fluorescently labeled oligonucleotides for reverse transcription reactions as primers, followed by the analysis of cDNA products using an automatic sequencer. This procedure allows the multiplexing and simultaneous analysis of a large number of samples. Over the past ten years, fluorescent toeprinting was applied to determine the activities of eukaryotic release factors and additional proteins involved in translation termination, to study the dynamics of translation initiation and elongation complexes, and to quantitatively evaluate the observed ribosomal complexes. Because of the simplicity and small amounts of material required, fluorescent toeprinting provides a highly scalable and versatile tool to study ribosomal complexes.


Assuntos
Bioensaio/métodos , Técnicas Genéticas , Ribossomos/metabolismo , Fluorescência , Células HeLa , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transcrição Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo
7.
Nucleic Acids Res ; 44(16): 7766-76, 2016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27418677

RESUMO

Poly(A)-binding protein (PABP) is a major component of the messenger RNA-protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl-tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1-eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.


Assuntos
Códon de Terminação/metabolismo , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Humanos , Hidrólise , Modelos Biológicos , Ligação Proteica , Aminoacil-RNA de Transferência
8.
Nucleic Acids Res ; 43(6): 3332-43, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25753665

RESUMO

Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNA(Val) and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.


Assuntos
Terminação Traducional da Cadeia Peptídica , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Acilação , Animais , Códon de Terminação , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Estabilidade Proteica , Estabilidade de RNA , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência de Valina/química , RNA de Transferência de Valina/genética , RNA de Transferência de Valina/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
BMC Mol Biol ; 10: 60, 2009 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-19545407

RESUMO

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation. RESULTS: To characterize further the functions of yeast eRF1 and eRF3, a genetic screen for their novel partner proteins was performed. As a result, the genes for gamma (TEF4 and TEF3/CAM1) and alpha (TEF5/EFB1) subunits of the translation elongation factor eEF1B, known to catalyze the exchange of bound GDP for GTP on eEF1A, were revealed. These genes act as dosage suppressors of a synthetic growth defect caused by some mutations in the SUP45 and SUP35 genes encoding eRF1 and eRF3, respectively. Extra copies of TEF5 and TEF3 can also suppress the temperature sensitivity of some sup45 and sup35 mutants and reduce nonsense codon readthrough caused by these omnipotent suppressors. Besides, overproduction of eEF1Balpha reduces nonsense codon readthrough in the strain carrying suppressor tRNA. Such effects were not shown for extra copies of TEF2, which encodes eEF1A, thus indicating that they were not due to eEF1A activation. CONCLUSION: The data obtained demonstrate involvement of the translation elongation factor eEF1B in modulating the functions of translation termination factors and suggest its possible role in GDP for GTP exchange on eRF3.


Assuntos
Terminação Traducional da Cadeia Peptídica , Fator 1 de Elongação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Saccharomyces cerevisiae/genética
10.
Proteomics Clin Appl ; 5(7-8): 432-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21751413

RESUMO

PURPOSE: The aim of this study was to estimate a possibility of mycosis fungoides (MF) diagnostics based on protein profiling in blood serum. EXPERIMENTAL DESIGN: We obtained and analysed samples of blood serum from 23 patients with MF, and 29 psoriasis patients and 22 healthy donors as controls. Protein profiling was carried out using SELDI TOF MS SELDI-TOF and also profiling of 27 cytokines with multiplex immunoassay technology was implemented. RESULTS: MS data analysis of sera did not give satisfactory statistical discrimination between the groups. Antibody-based cytokine profiling revealed a number of cytokines with a change in their concentrations in both MF and psoriasis (IL-1Ra, IL-4, G-CSF). The C-X-C motif chemokine 10 (IP-10, CXCL10) cytokine had a significantly increased concentration (p<0,001) in samples from MF patients as compared with the other groups. CONCLUSIONS AND CLINICAL RELEVANCE: IP-10 may be considered as a promising biomarker for the differentiation between MF and other skin conditions.


Assuntos
Biomarcadores/sangue , Quimiocina CXCL10/sangue , Imunoensaio/métodos , Micose Fungoide/diagnóstico , Proteoma/análise , Psoríase/diagnóstico , Neoplasias Cutâneas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Fator Estimulador de Colônias de Granulócitos/sangue , Humanos , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-4/sangue , Masculino , Pessoa de Meia-Idade , Micose Fungoide/sangue , Análise Serial de Proteínas , Psoríase/sangue , Pele , Neoplasias Cutâneas/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA