RESUMO
Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.
Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Motivos de Aminoácidos/genética , Animais , Códon de Terminação/genética , Euplotes/genética , Modelos Biológicos , Fatores de Terminação de Peptídeos/genética , Peptídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Supressão GenéticaRESUMO
In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Delta) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Delta mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5'-->3' pathway or the 3'-->5' nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Delta strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3' untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.
Assuntos
Fatores de Terminação de Peptídeos/metabolismo , RNA Fúngico/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas , Transporte Ativo do Núcleo Celular , Sequência de Bases , DNA Fúngico/genética , Genes Fúngicos , Modelos Biológicos , Mutação , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Fenótipo , Proteínas de Ligação a Poli(A)/metabolismo , Estabilidade de RNA , RNA Fúngico/genética , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.
Assuntos
Códon de Terminação , Euplotes/genética , Código Genético , Saccharomyces cerevisiae/genética , Tetrahymena thermophila/genética , Animais , Fatores de Terminação de Peptídeos/genética , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA de Transferência/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.
Assuntos
Códon de Terminação , Guanosina Trifosfato/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Sobrevivência Celular , GTP Fosfo-Hidrolases/metabolismo , Mutação , Fatores de Terminação de Peptídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The suppression of stop codons (termed translational readthrough) can be caused by a decreased accuracy of translation elongation or a reduced efficiency of translation termination. In previous studies, the inability to determine the extent to which each of these distinct processes contributes to a readthrough phenotype has limited our ability to evaluate how defects in the translational machinery influence the overall termination process. Here, we describe the combined use of misincorporation and readthrough reporter systems to determine which of these mechanisms contributes to translational readthrough in Saccharomyces cerevisiae. The misincorporation reporter system was generated by introducing a series of near-cognate mutations into functionally important residues in the firefly luciferase gene. These constructs allowed us to monitor the incidence of elongation errors by monitoring the level of firefly luciferase activity from a mutant allele inactivated by a single missense mutation. In this system, an increase in luciferase activity should reflect an increased level of misincorporation of the wild-type amino acid that provides an estimate of the overall fidelity of translation elongation. Surprisingly, we found that growth in the presence of paromomycin stimulated luciferase activity for only a small subset of the mutant proteins examined. This suggests that the ability of this aminoglycoside to induce elongation errors is limited to a subset of near-cognate mismatches. We also found that a similar bias in near-cognate misreading could be induced by the expression of a mutant form of ribosomal protein (r-protein) S9B or by depletion of r-protein L12. We used this misincorporation reporter in conjunction with a readthrough reporter system to show that alterations at different regions of the ribosome influence elongation fidelity and termination efficiency to different extents.
Assuntos
Regulação Fúngica da Expressão Gênica , Elongação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Saccharomyces cerevisiae/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sequência de Bases , Códon de Terminação/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Mutagênese/efeitos dos fármacos , Mutação/genética , Paromomicina/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fenótipo , RNA Helicases/deficiência , RNA Helicases/genética , RNA Helicases/metabolismo , RNA de Transferência/genética , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiaeRESUMO
The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was =0.5%. This low threshold for the onset of the major component of NMD indicates that mRNA surveillance is an ongoing process that occurs throughout the lifetime of an mRNA.