RESUMO
Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the 'hit rate' in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (DeltaG(o)37 < or = -30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide-RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (DeltaG(o)37) > or = -8 kcal/mol for inter-oligonucleotide pairing and (DeltaG(o)37) > or = -1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the 'hit rate' by as much as 6-fold.
Assuntos
Oligonucleotídeos Antissenso/química , Termodinâmica , Química Farmacêutica/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , Sensibilidade e EspecificidadeRESUMO
Optimization of probe design for array-based experiments requires improved predictability of oligonucleotide hybridization behavior. Currently, designing oligonucleotides capable of interacting efficiently and specifically with the relevant target is not a routine procedure. Multiple examples demonstrate that oligonucleotides targeting different regions of the same RNA differ in their hybridization ability. The present work shows how thermodynamic evaluations of oligo-target duplex or oligo self-structure stabilities can facilitate probe design. Statistical analysis of large sets of hybridization data reveals that thermodynamic evaluation of oligonucleotide properties can be used to avoid poor RNA binders. Thermodynamic criteria for the selection of 20 and 21mers, which, with high probability, interact efficiently and specifically with their targets, are suggested. The design of longer oligonucleotides can also be facilitated by the same calculations of DeltaG(o) (T) values for oligo-target duplex or oligo self-structure stabilities and similar selection schemes.
Assuntos
Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/genética , Hibridização de Ácido Nucleico , RNA/genética , Estatística como Assunto , TermodinâmicaRESUMO
BACKGROUND: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. RESULTS: HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3' deletion were more responsive to stimulation by polyamine addition than those with the 5' deletion. CONCLUSIONS: The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.
Assuntos
Mutação da Fase de Leitura , Regulação Enzimológica da Expressão Gênica/genética , Proteínas/genética , Animais , Sequência de Bases , Poliaminas Biogênicas/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , DNA , Eflornitina/farmacologia , Humanos , Dados de Sequência Molecular , Proteínas/antagonistas & inibidoresRESUMO
Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame. Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency. Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites. Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role. Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism.
Assuntos
Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , Ribossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófago T4/genética , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Proteínas de Transporte/genética , Códon/genética , Genes Virais/genética , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Ligação Proteica , RNA Bacteriano/genética , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/biossíntese , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing. Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.
Assuntos
Genes Virais/genética , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Anticódon/genética , Bacteriófago T4/genética , Pareamento de Bases , Sequência de Bases , Ligação Competitiva , Códon/genética , Escherichia coli/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Genótipo , Óperon Lac/genética , Espectrometria de Massas , Peso Molecular , Mutação/genética , Fases de Leitura Aberta/genética , Sinais Direcionadores de Proteínas/fisiologia , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/genética , Salmonella typhimurium/genéticaRESUMO
Bacterial tmRNA mediates a trans-translation reaction, which permits the recycling of stalled ribosomes and probably also contributes to the regulated expression of a subset of genes. Its action results in the addition of a small number of C-terminal amino acids to protein whose synthesis had stalled and these constitute a proteolytic recognition tag for the degradation of these incompletely synthesized proteins. Previous work has identified pseudoknots and stem-loops that are widely conserved in divergent bacteria. In the present work an alignment of tmRNA gene sequences within 13 beta-proteobacteria reveals an additional sub-structure specific for this bacterial group. This sub-structure is in pseudoknot Pk2, and consists of one to two additional stem-loop(s) capped by stable GNRA tetraloop(s). Three-dimensional models of tmRNA pseudoknot 2 (Pk2) containing various topological versions of the additional sub-structure suggest that the sub-structures likely point away from the core of the RNA, containing both the tRNA and the mRNA domains. A putative tertiary interaction has also been identified.
Assuntos
Betaproteobacteria/genética , Filogenia , RNA Bacteriano/genética , Sequência de Bases , DNA Bacteriano/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The RECODE database is a compilation of 'programmed' translational recoding events taken from the scientific literature and personal communications. The database deals with programmed ribosomal frameshifting, codon redefinition and translational bypass occurring in a variety of organisms. The entries for each event include the sequences of the corresponding genes, their encoded proteins for both the normal and alternate decoding, the types of the recoding events involved, trans-factors and cis-elements that influence recoding. The database is freely available at http://recode.genetics. utah.edu/.
Assuntos
Bases de Dados Factuais , Mudança da Fase de Leitura do Gene Ribossômico/genética , Sequência de Bases , Códon , Mutação da Fase de Leitura , Regulação da Expressão Gênica , Internet , Dados de Sequência MolecularAssuntos
Mutação da Fase de Leitura , Regulação da Expressão Gênica , Código Genético , Ribossomos/genética , Sequência de Bases , Modelos Genéticos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Ribossômico/química , RNA Ribossômico/genética , Proteínas Ribossômicas/genéticaRESUMO
SUMMARY: ODNBase is a database of antisense oligodeoxynucleotides targeted to mammalian mRNAs that were reported in the literature. It includes the oligo sequences tested, the measured effectiveness, the RNA that was targeted, the type of measurement assay used, the oligo concentration applied, and the reference for each oligo. It provides a searchable interface by motif content, activity level, applied concentration and RNA name. Oligo lists matching search criteria can be downloaded in a spreadsheet compatible format.
Assuntos
Bases de Dados Factuais , Desenho de Fármacos , Oligonucleotídeos Antissenso/genética , Animais , Humanos , Internet , Mamíferos , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , Interface Usuário-ComputadorAssuntos
Código Genético , Selenocisteína/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Códon , Códon de Terminação , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genéticaRESUMO
To expand the genetic code for specification of multiple non-natural amino acids, unique codons for these novel amino acids are needed. As part of a study of the potential of quadruplets as codons, the decoding of tandem UAGA quadruplets by an engineered tRNA(Leu) with an eight-base anticodon loop, has been investigated. When GCC is the codon immediately 5' of the first UAGA quadruplet, and release factor 1 is partially inactivated, the tandem UAGAs specify two leucines with an overall efficiency of at least 10%. The presence of a purine at anticodon loop position 32 of the tRNA decoding the codon 5' to the first UAGA seems to influence translation of the following codon. Another finding is intraribosomal dissociation of anticodons from codons and their re-pairing to mRNA at overlapping or nearby codons. In one case where GCC is replaced by CGG, only a single Watson-Crick base pair can form upon re-pairing when decoding is resumed. This has implications for the mechanism of some cases of programmed frameshifting.
Assuntos
Anticódon , Código Genético , RNA de Transferência de Leucina/genética , Códon , Escherichia coli/genética , Conformação de Ácido Nucleico , Proteínas/genética , RNA Mensageiro/genética , RNA de Transferência de Leucina/químicaRESUMO
Translational bypassing joins the information found within two disparate open reading frames into a single polypeptide chain. The underlying mechanism centers on the decoding properties of peptidyl-transfer RNA (tRNA) and involves three stages: take-off, scanning, and landing. In take-off, the peptidyl-tRNA/messenger RNA (mRNA) complex in the P site of the ribosome dissociates, and the mRNA begins to move through the ribosome. In scanning, the peptidyl-tRNA probes the mRNA sliding through the decoding center. In landing, the peptidyl-tRNA re-pairs with a codon with which it can form a stable interaction. Although few examples of genes are known that rely on translational bypassing to couple open reading frames, ribosomes appear to have an innate capacity for bypassing. This suggests that the strategy of translational bypassing may be more common than presently appreciated. The best characterized example of this phenomenon is T4 gene 60, in which a complex set of signals stimulates bypassing of 50 nucleotides between the two open reading frames. In this review, we focus on the bypassing mechanism of gene 60 in terms of take-off, scanning, and landing.
Assuntos
Fases de Leitura Aberta , Biossíntese de Proteínas , Sequência de Aminoácidos , Sequência de Bases , Genes Bacterianos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Terminação Traducional da Cadeia Peptídica , Sinais Direcionadores de Proteínas/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
The efficiency of programmed ribosomal frameshifting in decoding antizyme mRNA is the sensor for an autoregulatory circuit that controls cellular polyamine levels in organisms ranging from the yeast Schizosaccharomyces pombe to Drosophila to mammals. Comparison of the frameshift sites and flanking stimulatory signals in many organisms now permits a reconstruction of the likely evolutionary path of the remarkably conserved mRNA sequences involved in the frameshifting.
Assuntos
Códon/genética , Sequência Conservada/genética , Evolução Molecular , Mudança da Fase de Leitura do Gene Ribossômico/genética , Poliaminas/metabolismo , RNA Catalítico/genética , Animais , Sequência de Bases , Retroalimentação , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Alinhamento de SequênciaRESUMO
As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside-induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside-induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside-induced readthrough in the order C > U > A > or = G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33-base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low-level aminoglycoside-induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed.
Assuntos
Antibacterianos/farmacologia , Códon de Terminação/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Células Cultivadas , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Distrofina/genética , Gentamicinas/farmacologia , Humanos , Camundongos , Distrofia Muscular de Duchenne/fisiopatologia , Mutação/efeitos dos fármacos , Mutação/genética , Biossíntese de Proteínas/genética , Fases de Leitura/genéticaRESUMO
Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent.
Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Composição de Bases , Sequência de Bases , Citosina , Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonuclease H/metabolismo , Relação Estrutura-AtividadeRESUMO
Translating ribosomes bypass a 50 nt coding gap in order to fuse the information found in the two open reading frames (ORFs) of bacteriophage T4 gene 60. This study investigates the underlying mechanism by focusing on the competition between initiation of bypassing and termination at the end of the first ORF. While nearly all ribosomes initiate bypassing, no more than 50% resume translation in the second ORF. Two previously described cis-acting stimulatory signals are critical for favoring initiation of bypassing over termination. Genetic analysis of these signals supports a working model in which the first (a stem-loop structure at the junction between the first ORF and the coding gap) interferes with decoding in the A-site, and the second (a stretch of amino acids in the nascent peptide encoded by the first ORF) destabilizes peptidyl-tRNA-mRNA pairing.
Assuntos
Bacteriófago T4/genética , Fases de Leitura Aberta , Biossíntese de Proteínas , Ribossomos/fisiologia , Sequência de Aminoácidos , Bacteriófago T4/metabolismo , Sequência de Bases , Códon/genética , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/metabolismo , RNA de Transferência de Glicina/genéticaRESUMO
Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.
Assuntos
Mutação da Fase de Leitura , Poliaminas/metabolismo , Proteínas/química , Schizosaccharomyces/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Sequência Conservada , Evolução Molecular , Deleção de Genes , Humanos , Dados de Sequência Molecular , Mutagênese , Biossíntese de Proteínas , Proteínas/genética , Putrescina/metabolismo , Homologia de Sequência de Aminoácidos , Espermidina/metabolismo , Espermina/metabolismo , Transcrição GênicaRESUMO
Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.
Assuntos
Escherichia coli/química , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sítios de Ligação , Ácido Edético/metabolismo , Escherichia coli/genética , Compostos Ferrosos/metabolismo , Engenharia Genética , Radical Hidroxila/metabolismo , Modelos Moleculares , Sondas Moleculares/metabolismo , Peso Molecular , Mutação/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/genética , Ésteres do Ácido Sulfúrico/metabolismoRESUMO
One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid. The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined. With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 %. The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations. The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA. When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced. Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet.
