RESUMO
Plants encode subunits for a fourth RNA polymerase (Pol IV) in addition to the well-known DNA-dependent RNA polymerases I, II, and III. By mutation of the two largest subunits (NRPD1a and NRPD2), we show that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3. The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.
Assuntos
Arabidopsis/enzimologia , DNA de Plantas/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Inativação Gênica , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Cromatina/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis , DNA de Plantas/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Genes de Plantas , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Mutação , Oryza/enzimologia , Oryza/genética , Plantas Geneticamente Modificadas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , RNA de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Sequências Repetitivas de Ácido Nucleico , TransgenesRESUMO
Small RNAs serve as the specificity determinant for a collection of regulatory mechanisms known as RNA silencing. Plants use these mechanisms to control the expression of endogenous genes and to suppress unwanted foreign nucleic acids. Several gene families implicated in silencing have undergone expansion and evidence exists for multiple RNA silencing pathways. Recent progress in defining the components of a number of these pathways is examined here.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/genética , Metilação de DNA , Proteínas Fúngicas/genética , Plantas/genética , RNA de Cadeia Dupla/metabolismo , RNA de Plantas/genética , Ribonuclease III/metabolismoRESUMO
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/genéticaAssuntos
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
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
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
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
Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein. Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA2Gly. Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency. Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing.
Assuntos
Bacteriófago T4/genética , Biossíntese de Proteínas , RNA de Transferência de Glicina/genética , Pareamento de Bases , Sequência de Bases , Escherichia coli/genética , Genes Virais , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Mensageiro/genética , Salmonella typhimurium/genética , Proteínas Virais/genéticaRESUMO
Ribosomes translating bacteriophage T4 gene 60 mRNA bypass 50 noncoding nucleotides from a takeoff site at codon 46 to a landing site just upstream of codon 47. A key signal for efficient bypassing is contained within the nascent peptide synthesized prior to takeoff. Here we show that this signal is insensitive to the addition of coding information at its N terminus. In addition, analysis of amino-terminal fusions, which allow detection of all major products synthesized from the gene 60 mRNA, show that 50% of ribosomes bypass the coding gap while the rest either terminate at a UAG stop codon immediately following codon 46 or fail to resume coding. Bypassing efficiency estimates significantly lower than 50% were obtained with enzymatic reporter systems that relied on comparing test constructs to constructs with a precise excision of the gap (gap deletion). Further analysis showed that these estimates are distorted by differences between test and gap deletion functional mRNA levels. An internal translation initiation site at Met12 of gene 60 (which eliminates part of the essential nascent peptide) also distorts these estimates. Together, these results support an efficiency estimate of approximately 50%, less than previously reported. This estimate suggests that bypassing efficiency is determined by the competition between reading signals and release factors and gives new insight into the kinetics of bypassing signal action.