RESUMO
RNAs somehow adopt specific functional structures despite the capacity to form alternative nonfunctional structures with similar stabilities. We analyzed RNA assembly during transcription in vitro and in yeast using hairpin ribozyme self-cleavage to assess partitioning between functional ribozyme structures and nonfunctional stem loops. Complementary insertions located upstream of the ribozyme inhibited ribozyme assembly more than downstream insertions during transcription in vitro, consistent with a sequential folding model in which the outcome is determined by the structure that forms first. In contrast, both upstream and downstream insertions strongly inhibited assembly of the same ribozyme variants when expressed as chimeric mRNAs in yeast, indicating that inhibitory stem loops can form even after the entire ribozyme sequence has been transcribed. Evidently, some feature unique to the intracellular environment modulates the influence of transcription polarity and enhances the contribution of thermodynamic stability to RNA folding in vivo.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Fúngico/química , Saccharomyces cerevisiae/metabolismo , Termodinâmica , Genes Fúngicos , Variação Genética , Técnicas In Vitro , Cinética , Modelos Biológicos , Mutação , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Fúngico/análise , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Temperatura , Transcrição GênicaRESUMO
The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.
Assuntos
Códon sem Sentido , Mutação da Fase de Leitura , Proteínas Fúngicas/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Códon de Terminação , Escherichia coli/genética , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos , Genes Reporter , Óperon Lac , Luciferases/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Helicases/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genéticaRESUMO
Increased efficiencies of programmed -1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA "Killer" virus. Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased peptidyltransferase activities. The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay. Independent experiments demonstrate similar programmed -1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41, which has previously been shown to result in peptidyltransferase defects in yeast. These findings are consistent with the hypothesis that decreased peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle, allowing more time for ribosomal slippage at programmed -1 ribosomal frameshift signals.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Peptidil Transferases/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Dados de Sequência Molecular , Plasmídeos , RNA Mensageiro/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/virologia , Homologia de Sequência de AminoácidosRESUMO
A new in vivo assay system has been developed to study programmed frameshifting in the yeast Saccharomyces cerevisiae. Frameshift signals are inserted between the Renilla and firefly luciferase reporter genes contained in a yeast expression vector and the two activities are directly measured from cell lysates in one tube. Similar to other bicistronic reporter systems, this one allows the efficient estimation of recoding efficiency by comparison of the normalized activity ratios from each luciferase protein. The assay system has been applied to HIV-1 and L-A directed programmed -1 frameshifting and Ty1 and Ty3 directed +1 frameshifting. The assay system is amenable to high-throughput screening.
Assuntos
Luciferases/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Animais , Sequência de Bases , Besouros , Mudança da Fase de Leitura do Gene Ribossômico , Genes Reporter , Dados de Sequência Molecular , RNA Mensageiro/genéticaRESUMO
There is something special about mRNA pseudoknots that allows them to elicit efficient levels of programmed -1 ribosomal frameshifting. Here, we present a synthesis of recent crystallographic, molecular, biochemical, and genetic studies to explain this property. Movement of 9 A by the anticodon loop of the aminoacyl-tRNA at the accommodation step normally pulls the downstream mRNA a similar distance along with it. We suggest that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel. These two opposing forces result in the creation of a local region of tension in the mRNA between the A-site codon and the mRNA pseudoknot. This can be relieved by one of two mechanisms; unwinding the pseudoknot, allowing the downstream region to move forward, or by slippage of the proximal region of the mRNA backwards by one base. The observed result of the latter mechanism is a net shift of reading frame by one base in the 5' direction, that is, a -1 ribosomal frameshift.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Bactérias/química , Bactérias/metabolismo , TermodinâmicaRESUMO
Many viral mRNAs, including those of HIV-1, can make translating ribosomes change reading frame. Altering the efficiencies of programmed ribosomal frameshift (PRF) inhibits viral propagation. As a new target for potential antiviral agents, it is therefore important to understand how PRF is controlled. Incorporation of the current models describing PRF into the context of the translation elongation cycle leads us to propose an 'integrated model' of PRF both as a guide towards further characterization of PRF at the molecular and biochemical levels, and for the identification of new targets for antiviral therapeutics.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/fisiologia , Fases de Leitura , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/fisiologiaRESUMO
Many viruses use programmed -1 ribosomal frameshifting to ensure the correct ratio of viral structural to enzymatic proteins. Alteration of frameshift efficiencies changes these ratios, in turn inhibiting viral particle assembly and virus propagation. Previous studies determined that anisomycin, a peptidyl transferase inhibitor, specifically inhibited -1 frameshifting and the ability of yeast cells to propagate the L-A and M(1) dsRNA viruses (J. D. Dinman, M. J. Ruiz-Echevarria, K. Czaplinski, and S. W. Peltz, 1997, Proc. Natl. Acad. Sci. USA 94, 6606-6611). Here we show that preussin, a pyrollidine that is structurally similar to anisomycin (R. E. Schwartz, J. Liesch, O. Hensens, L. Zitano, S. Honeycutt, G. Garrity, R. A. Fromtling, J. Onishi, and R. Monaghan, 1988. J. Antibiot. (Tokyo) 41, 1774--1779), also inhibits -1 programmed ribosomal frameshifting and virus propagation by acting at the same site or through the same mechanism as anisomycin. Since anisomycin is known to assert its effect at the ribosomal A-site, we undertook a pharmacogenetic analysis of mutants of trans-acting eukaryotic elongation factors (eEFs) that function at this region of the ribosome. Among mutants of eEF1A, a correlation is observed between resistance/susceptibility profiles to preussin and anisomycin, and these in turn correlate with programmed -1 ribosomal frameshifting efficiencies and killer virus phenotypes. Among mutants of eEF2, the extent of resistance to preussin correlates with resistance to sordarin, an eEF2 inhibitor. These results suggest that structural features associated with the ribosomal A-site and with the trans-acting factors that interact with it may present a new set of molecular targets for the rational design of antiviral compounds.
