共查询到20条相似文献,搜索用时 15 毫秒
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A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting 总被引:2,自引:0,他引:2
Programmed ribosomal frameshifting (PRF) is a process by which ribosomes produce two different polypeptides from the same mRNA. In this study, we propose three different kinetic models of +1 PRF, incorporating the effects of the ribosomal E-, P- and A-sites toward promoting efficient +1 frameshifting in Escherichia coli. Specifically, the timing of E-site tRNA dissociation is discussed within the context of the kinetic proofreading mechanism of aminoacylated tRNA (aa-tRNA) selection. Mathematical modeling using previously determined kinetic rate constants reveals that destabilization of deacylated tRNA in the E-site, rearrangement of peptidyl-tRNA in the P-site, and availability of cognate aa-tRNA corresponding to the A-site act synergistically to promote efficient +1 PRF. The effect of E-site codon:anticodon interactions on +1 PRF was also experimentally examined with a dual fluorescence reporter construct. The combination of predictive modeling and empirical testing allowed the rate constant for P-site tRNA slippage (ks) to be estimated as ks ≈1.9 s−1 for the release factor 2 (RF2) frameshifting sequence. These analyses suggest that P-site tRNA slippage is the driving force for +1 ribosomal frameshifting while the presence of a ‘hungry codon’ in the A-site and destabilization in the E-site further enhance +1 PRF in E. coli. 相似文献
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The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the ~70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine–Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure. 相似文献
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Does Disparate Occurrence of Autoregulatory Programmed Frameshifting in Decoding the Release Factor 2 Gene Reflect an Ancient Origin with Loss in Independent Lineages? 总被引:1,自引:0,他引:1 下载免费PDF全文
In Escherichia coli an autoregulatory mechanism of programmed ribosomal frameshifting governs the level of polypeptide chain release factor 2. From an analysis of 20 sequences of genes encoding release factor 2, we infer that this frameshift mechanism was present in a common ancestor of a large group of bacteria and has subsequently been lost in three independent lineages. 相似文献
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Betty Y.-W. Chung 《Journal of molecular biology》2010,397(2):448-456
Programmed ribosomal frameshifting allows the synthesis of alternative, N-terminally coincident, C-terminally distinct proteins from the same RNA. Many viruses utilize frameshifting to optimize the coding potential of compact genomes, to circumvent the host cell's canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed ratio. Programmed frameshifting is also used in the decoding of a small number of cellular genes. Recently, specific ribosomal − 1 frameshifting was discovered at a conserved U_UUU_UUA motif within the sequence encoding the alphavirus 6K protein. In this case, frameshifting results in the synthesis of an additional protein, termed TF (TransFrame). This new case of frameshifting is unusual in that the − 1 frame ORF is very short and completely embedded within the sequence encoding the overlapping polyprotein.The present work shows that there is remarkable diversity in the 3′ sequences that are functionally important for efficient frameshifting at the U_UUU_UUA motif. While many alphavirus species utilize a 3′ RNA structure such as a hairpin or pseudoknot, some species (such as Semliki Forest virus) apparently lack any intra-mRNA stimulatory structure, yet just 20 nt 3′-adjacent to the shift site stimulates up to 10% frameshifting. The analysis, both experimental and bioinformatic, significantly expands the known repertoire of − 1 frameshifting stimulators in mammalian and insect systems. 相似文献
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Virag Sharma Marie-Fran?oise Prère Isabelle Canal Andrew E. Firth John F. Atkins Pavel V. Baranov Olivier Fayet 《Nucleic acids research》2014,42(11):7210-7225
Programmed ribosomal -1 frameshifting is a non-standard decoding process occurring when ribosomes encounter a signal embedded in the mRNA of certain eukaryotic and prokaryotic genes. This signal has a mandatory component, the frameshift motif: it is either a Z_ZZN tetramer or a X_XXZ_ZZN heptamer (where ZZZ and XXX are three identical nucleotides) allowing cognate or near-cognate repairing to the -1 frame of the A site or A and P sites tRNAs. Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%. The present study combines experimental and bioinformatics approaches to carry out (i) a systematic analysis of the frameshift propensity of all possible motifs (16 Z_ZZN tetramers and 64 X_XXZ_ZZN heptamers) in Escherichia coli and (ii) the identification of genes potentially using this mode of expression amongst 36 Enterobacteriaceae genomes. While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ. The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting. 相似文献
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Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor. 相似文献
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Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo 总被引:1,自引:0,他引:1
Mora L Heurgué-Hamard V de Zamaroczy M Kervestin S Buckingham RH 《The Journal of biological chemistry》2007,282(49):35638-35645
Bacterial release factors RF1 and RF2 are methylated on the Gln residue of a universally conserved tripeptide motif GGQ, which interacts with the peptidyl transferase center of the large ribosomal subunit, triggering hydrolysis of the ester bond in peptidyl-tRNA and releasing the newly synthesized polypeptide from the ribosome. In vitro experiments have shown that the activity of RF2 is stimulated by Gln methylation. The viability of Escherichia coli K12 strains depends on the integrity of the release factor methyltransferase PrmC, because K12 strains are partially deficient in RF2 activity due to the presence of a Thr residue at position 246 instead of Ala. Here, we study in vivo RF1 and RF2 activity at termination codons in competition with programmed frameshifting and the effect of the Ala-246 --> Thr mutation. PrmC inactivation reduces the specific termination activity of RF1 and RF2(Ala-246) by approximately 3- to 4-fold. The mutation Ala-246 --> Thr in RF2 reduces the termination activity in cells approximately 5-fold. After correction for the decrease in level of RF2 due to the autocontrol of RF2 synthesis, the mutation Ala-246 --> Thr reduced RF2 termination activity by approximately 10-fold at UGA codons and UAA codons. PrmC inactivation had no effect on cell growth in rich media but reduced growth considerably on poor carbon sources. This suggests that the expression of some genes needed for optimal growth under such conditions can become growth limiting as a result of inefficient translation termination. 相似文献
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Translational misreading: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshifting and drug sensitivity. 总被引:4,自引:0,他引:4 下载免费PDF全文
The translation elongation feactor 1alpha (EF-1alpha) catalyzes the critical step of delivering aminoacyl-tRNAs to the elongating ribosome. A series of Saccharomyces cerevisiae strains containing mutant alleles of the TEF2 gene encoding EF-1alpha have phenotypes consistent with effects on cellular processes related to translation. These include (1) conditional growth defects, (2) antibiotic sensitivity or resistance, (3) altered +1 or -1 ribosomal frameshifting efficiencies, and (4) altered maintenance of the killer phenotype. Although all the mutant alleles were isolated as dominant +1 frameshift suppressors, the effects of these mutations on the cell are quite different when present as the only form of EF-1alpha. Allele-specific effects are observed with regard to their ability to alter the efficiency of programmed +1 frameshifting as opposed to programmed -1 ribosomal frameshifting. The significantly altered efficiency of -1 frameshifting in strains containing the TEF2-4 and TEF2-9 mutant alleles further correlates with a reduced ability to maintain the killer phenotype and the M1 satellite virus of L-A, an in vivo assay of translational fidelity. In light of the proposed models regarding the different A- and P-site occupancy states required for +1 or -1 ribosomal frameshifting, these results aid analysis of interactions between EF-1alpha and the translational apparatus. 相似文献
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Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2 总被引:7,自引:0,他引:7
Maintenance of the translation reading frame is one of the most remarkable achievements of the ribosome while decoding the information of an mRNA. Loss of the reading frame through spontaneous frameshifting occurs with a frequency of one in 30,000 amino acid incorporations. However, at many recoding sites, the mechanism that controls reading frame maintenance is switched off. One such example is the programmed +1 frameshift site of the prfB gene encoding the termination factor RF2, in which slippage into the forward frame by one nucleotide can attain an efficiency of approximately 100%, namely, four orders of magnitude higher than normally observed. Here, using the RF2 frameshift window, we demonstrate that premature release of the E site tRNA from the ribosome is coupled with high-level frameshifting. Consistently, in a minimal system, the presence of the E site tRNA prevents the +1 frameshift event, illustrating the importance of the E site for reading-frame maintenance. 相似文献
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Recognizing the pseudogenes in bacterial genomes 总被引:9,自引:0,他引:9
Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1–8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as ‘hypothetical’ or ‘unknown’; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes. 相似文献
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Antisense-induced ribosomal frameshifting 总被引:1,自引:0,他引:1
Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels. 相似文献
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In many viruses, -1 ribosomal frameshifting (-1RF) regulates synthesis of proteins and is crucial for virus production. An RNA pseudoknot is one of the essential components of the viral -1RF system. Thermodynamic or kinetic control of pseudoknot folding may be important in regulating the efficiency of -1RF. Thus, small molecules that interact with viral RNA pseudoknots may disrupt the -1RF system and show antiviral activity. In this study, we conducted virtual screening of a chemical database targeting the X-ray crystal structure of RNA pseudoknot complexed with biotin to identify ligands that may regulate an -1RF system containing biotin-aptamer as an RNA pseudoknot component. After docking screening of about 80,000 compounds, 58 high-ranked hits were selected and their activities were examined by in vitro and cell-based -1 frameshifting assays. Six compounds increased the efficiency of -1 frameshifting, and these are novel small molecule compounds that regulate the -1RF. 相似文献
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Phylogenomics of prokaryotic ribosomal proteins 总被引:1,自引:0,他引:1
Archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). Despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. We developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 archaeal genomes available in the RefSeq database. The pipeline employs curated seed alignments of r-proteins to run position-specific scoring matrix (PSSM)-based BLAST searches against six-frame genome translations, mitigating possible gene annotation errors. As a result of this analysis, we performed a census of prokaryotic r-protein complements, enumerated missing and paralogous r-proteins, and analyzed the distributions of ribosomal protein genes among chromosomal partitions. Phyletic patterns of bacterial and archaeal r-protein genes were mapped to phylogenetic trees reconstructed from concatenated alignments of r-proteins to reveal the history of likely multiple independent gains and losses. These alignments, available for download, can be used as search profiles to improve genome annotation of r-proteins and for further comparative genomics studies. 相似文献
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