RESUMO
The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteínas Virais/metabolismo , Ribossomos/metabolismo , Theilovirus/genética , Theilovirus/metabolismo , Proteínas Virais/químicaRESUMO
Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a 'slippery' sequence and a 3'-adjacent RNA structure. Recently, we showed that -1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Viral/genética , Ribossomos/genética , Theilovirus/genética , Animais , Sequência de Bases , Camundongos , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Ribossomos/metabolismo , Theilovirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The -2/-1 programmed ribosomal frameshifting (-2/-1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This -2/-1 PRF mechanism is transactivated by a viral protein, nsp1ß, and cellular poly(rC) binding proteins (PCBPs). Critical elements for -2/-1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate -2 PRF to generate nsp2TF. The nsp1ß of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both -2 and -1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1ß are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus -2/-1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1ß, slippery sequence, and C-rich motif in -2/-1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of -2/-1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, -2/-1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1ßs of all non-EAV arteriviruses tested. Taken together, these data suggest that -2/-1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication.IMPORTANCE Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved ribosomal frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.
Assuntos
Evolução Biológica , Mudança da Fase de Leitura do Gene Ribossômico , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arterivirus/genética , Linhagem Celular , Expressão Gênica , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide 'slippery' sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both -2 and -1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1ß. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1ß and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1ß complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus-host interactions.
Assuntos
Cisteína Endopeptidases/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , RNA Mensageiro/genética , Ribossomos/genética , Animais , Sequência de Bases , Cisteína Endopeptidases/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Biossíntese de Proteínas/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/genética , Suínos/genética , Suínos/virologiaRESUMO
Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1ß). Embedded in nsp1ß's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1ß with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Regulação Viral da Expressão Gênica/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Ativação Transcricional/fisiologia , Proteínas não Estruturais Virais/fisiologia , Animais , Linhagem Celular , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Haplorrinos , Humanos , Imunoensaio , Luciferases , Corantes de Rosanilina , Espectrometria de Massas em TandemRESUMO
Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed-1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that-1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3= RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient-1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Theilovirus/genética , Animais , Linhagem Celular , Immunoblotting , Luciferases , Espectrometria de Massas , Camundongos , Mutagênese , Recombinação Genética/genética , Corantes de Rosanilina , Ensaio de Placa ViralRESUMO
Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4⺠T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4⺠T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por Caliciviridae/imunologia , Proteínas do Capsídeo/imunologia , Memória Imunológica , Norovirus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/prevenção & controle , Proteínas do Capsídeo/genética , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Humanos , Camundongos , Camundongos Knockout , Norovirus/genética , Especificidade da Espécie , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.
Assuntos
Códon de Terminação/genética , Vírus da Febre do Carrapato do Colorado/genética , Terminação Traducional da Cadeia Peptídica/genética , RNA/genética , Animais , Sequência de Bases , Sistema Livre de Células , Códon de Terminação/metabolismo , Vírus da Febre do Carrapato do Colorado/metabolismo , Dermacentor/genética , Dermacentor/metabolismo , Insetos/genética , Insetos/metabolismo , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas/genética , RNA/metabolismo , Ribossomos/metabolismoRESUMO
The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.
Assuntos
Códon de Terminação/genética , Coronavirus/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Escherichia coli/genética , Regulação Viral da Expressão Gênica , Humanos , Vírus da Leucemia Murina/genética , Proteínas com Motivo de Reconhecimento de RNA , RNA Viral/genética , Replicação ViralRESUMO
Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
Assuntos
Infecções por Cardiovirus/virologia , Vírus da Encefalomiocardite/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Virais/química , Proteínas Virais/metabolismo , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/metabolismo , Cristalografia por Raios X , Vírus da Encefalomiocardite/química , Vírus da Encefalomiocardite/genética , Mudança da Fase de Leitura do Gene Ribossômico , Humanos , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Virais/genéticaRESUMO
Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.
Assuntos
Vírus da Influenza B/metabolismo , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Proteínas Virais/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Códon de Terminação/genética , Códon de Terminação/metabolismo , Vírus da Influenza B/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genéticaRESUMO
Programmed -1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins.
Assuntos
Vírus da Encefalomiocardite/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Regulação Viral da Expressão Gênica/genética , Sequências Repetidas Invertidas/genética , Biossíntese de Proteínas/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Vírus da Encefalomiocardite/genética , Mesocricetus , Conformação de Ácido Nucleico , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Viral/biossíntese , RNA Viral/genética , Ribossomos/metabolismoRESUMO
BACKGROUND: Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAAUG. PRINCIPAL FINDINGS: Here, we confirm that MNV VP2 expression is regulated by termination-reinitiation and define the mRNA sequence requirements. Efficient reintiation is dependent upon 43 nt of RNA immediately upstream of the UAAUG site. Chemical and enzymatic probing revealed that the RNA in this region is not highly structured and includes an essential stretch of bases complementary to 18S rRNA helix 26 (Motif 1). The relative position of Motif 1 with respect to the UAAUG site impacts upon the efficiency of the process. Termination-reinitiation in MNV was also found to be relatively insensitive to the initiation inhibitor edeine. CONCLUSIONS: The termination-reinitiation signal of MNV most closely resembles that of influenza BM2. Similar to other viruses that use this strategy, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the VP1 stop codon. Our data also indicate that accurate recognition of the VP2 ORF AUG is not a pre-requisite for efficient reinitiation of translation in this system.
Assuntos
Norovirus/metabolismo , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Proteínas Virais/metabolismo , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Códon de Iniciação/genética , Códon de Terminação/genética , Edeína/farmacologia , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Norovirus/efeitos dos fármacos , Conformação de Ácido Nucleico , Nucleotídeos/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Complementar/genética , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , RNA Viral/química , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.
Assuntos
Expressão Gênica , Lisina/análogos & derivados , Biossíntese de Proteínas , Animais , Sequência de Bases , Códon , Escherichia coli/genética , Humanos , Lisina/química , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Selenocisteína/químicaRESUMO
Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.