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End-to-end RNA sequencing methods that capture 5'-sequence content without cumbersome library manipulations are of great interest, particularly for analysis of long RNAs. While template-switching methods have been developed for RNA sequencing by distributive short-read RTs, such as the MMLV RT enzymes used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those that derive from group II self-splicing introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multi-step template switching reaction carried out by RT enzymes, in this case, by a well-characterized enzyme known as MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized and optimized the enzymatic nontemplated addition (NTA) reaction that occurs when the RT enzyme extends past the RNA 5'-terminus, and we determined the nucleotide specificity of the NTA reaction. We then evaluated the binding specificity of specialized template-switching oligonucleotides, optimizing their sequences and chemical properties to guide efficient template switching reaction. Having dissected and optimized these individual steps, we then unified them into a procedure for performing RNA sequencing with MarathonRT enzymes, using a well-characterized RNA reference set. The resulting reads span a six-log range in transcript concentration and accurately represent the input RNA identities in both length and composition. We also performed RNA-seq starting from total human RNA and poly(A)-enriched RNA, with short and long-read sequencing demonstrating that MarathonRT enhances the discovery of unseen RNA molecules by conventional RT. Altogether, by employing mechanistic enzymology on RT enzymes and using them to modify RNA-seq technologies, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.
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Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.
Assuntos
Biotecnologia , DNA Polimerase Dirigida por RNA , Análise de Sequência de RNA , DNA Polimerase Dirigida por RNA/genética , Análise de Sequência de RNA/métodosRESUMO
The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRSâ¢tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.
Assuntos
Aminoacil-tRNA Sintetases , Archaea , Código Genético , Lisina , Aminoacil-tRNA Sintetases/metabolismo , Archaea/enzimologia , Archaea/genética , Lisina/análogos & derivados , Lisina/genética , Methanosarcina , RNA de Transferência/genéticaRESUMO
This corrects the article DOI: 10.1038/nchembio.2474.
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Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation.
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Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Escherichia coli/biossíntese , Escherichia coli/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Canamicina/farmacologia , Espectrometria de Massas , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Estresse Oxidativo/genética , Peroxirredoxinas/biossíntese , Peroxirredoxinas/genética , Dobramento de Proteína , Proteômica/métodos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Estreptomicina/farmacologia , Fatores de TempoRESUMO
Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.
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Aminoacil-tRNA Sintetases/metabolismo , Evolução Molecular Direcionada , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Biocatálise , Methanocaldococcus/metabolismo , Methanosarcina/metabolismo , Conformação Molecular , Proteínas/química , Proteínas/metabolismoRESUMO
Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution-a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Lisina/análogos & derivados , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Cristalografia por Raios X , Evolução Molecular Direcionada , Lisina/química , Lisina/metabolismo , Methanosarcina/enzimologia , Modelos MolecularesRESUMO
Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRSâ¢Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.
Assuntos
Alanina/análogos & derivados , Staphylococcus aureus , Tiofenos/química , Tiorredoxinas/química , Triptofano/química , Alanina/química , Alanina/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Domínio Catalítico , Cinética , Modelos Moleculares , Sondas Moleculares , Engenharia de Proteínas , Staphylococcus aureus/genética , Tiofenos/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.
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Aminoacil-tRNA Sintetases/metabolismo , Evolução Molecular Direcionada , Lisina/metabolismo , CinéticaRESUMO
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by inflammatory cell infiltration, synovial inflammation, and cartilage destruction. Proliferative fibroblast-like synoviocytes (FLS) play crucial roles in both propagation of inflammation and joint damage because of their production of great amount of proinflammatory cytokines and proteolytic enzymes. In this study, we investigate the role of TRAF-interacting protein (TRIP) in regulating inflammatory process in RA-FLS. TRIP expression was attenuated in RA-FLS compared with osteoarthritis- (OA-) FLS. Overexpression of TRIP significantly inhibited the activation of NF-κB signaling and decreased the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) in TNFα-stimulated RA-FLS. Furthermore, TRIP was found to interact with transforming growth factor ß-activated kinase 1 (TAK1) and promoting K48-linked polyubiquitination of TAK1 in RA-FLS. Our results demonstrate that TRIP has anti-inflammatory effects on RA-FLS and suggest TRIP as a potential therapeutic target for human RA.
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Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Osteoartrite/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lentivirus , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Líquido Sinovial/metabolismoRESUMO
Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.
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Aminoacil-tRNA Sintetases/química , Glutamato-tRNA Ligase/química , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Domínio Catalítico , Evolução Molecular Direcionada , Escherichia coli/enzimologia , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Ácido Glutâmico/metabolismo , Helicobacter pylori/enzimologia , Dados de Sequência Molecular , Engenharia de Proteínas , RNA de Transferência de Glutamina/metabolismo , Alinhamento de Sequência , Temperatura , Aminoacilação de RNA de TransferênciaRESUMO
Leifsonia aquatica is an aquatic bacterium that is typically found in environmental water habitats. Infections due to L. aquatica are rare and commonly catheter associated in immunocompromised patients. We report the first case of an acute septicemia caused by L. aquatica in a healthy immunocompetent host after cryopexy in the absence of a catheter.
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Infecções por Actinomycetales/diagnóstico , Actinomycetales/isolamento & purificação , Complicações Pós-Operatórias/diagnóstico , Retina/cirurgia , Sepse/diagnóstico , Infecções por Actinomycetales/microbiologia , Infecções por Actinomycetales/patologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Complicações Pós-Operatórias/microbiologia , Complicações Pós-Operatórias/patologia , RNA Ribossômico 16S/genética , Sepse/microbiologia , Sepse/patologia , Análise de Sequência de DNARESUMO
Although next-generation sequencing (NGS) technologies have revolutionized our ability to sequence DNA with high-throughput, the chain termination-based Sanger sequencing method remains a widely used approach for DNA sequence analysis due to its simplicity, low cost and high accuracy. In particular, high accuracy makes Sanger sequencing the "gold standard" for sequence validation in basic research and clinical applications. During the early days of Sanger sequencing development, reverse transcriptase (RT)-based RNA sequencing was also explored and showed great promise, but the approach did not acquire popularity over time due to the limited processivity and low template unwinding capability of Avian Myeloblastosis Virus (AMV) RT, and other RT enzymes available at the time. RNA molecules have complex features, often containing repetitive sequences and stable secondary or tertiary structures. While these features are required for RNA biological function, they represent strong obstacles for retroviral RTs. Repetitive sequences and stable structures cause reverse transcription errors and premature primer extension stops, making chain termination-based methods unfeasible. MarathonRT is an ultra-processive RT encoded group II intron that can copy RNA molecules of any sequence and structure in a single cycle, making it an ideal RT enzyme for Sanger RNA sequencing. In this chapter, we upgrade the Sanger RNA sequencing method by replacing AMV RT with MarathonRT, providing a simple, robust method for direct RNA sequence analysis. The guidance for troubleshooting and further optimization are also provided.
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DNA Polimerase Dirigida por RNA , RNA , RNA/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNARESUMO
RNA molecules play important roles in numerous normal cellular processes and disease states, from protein coding to gene regulation. RT-PCR, applying the power of polymerase chain reaction (PCR) to RNA by coupling reverse transcription with PCR, is one of the most important techniques to characterize RNA transcripts and monitor gene expression. The ability to analyze full-length RNA transcripts and detect their expression is critical to decipher their biological functions. However, due to the low processivity of retroviral reverse transcriptases (RTs), we can only monitor a small fraction of long RNA transcripts, especially those containing stable secondary and tertiary structures. The full-length sequences can only be deduced by computational analysis, which is often misleading. Group II intron-encoded RTs are a new type of RT enzymes. They have evolved specialized structural elements that unwind template structures and maintain close contact with the RNA template. Therefore, group II intron-encoded RTs are more processive than the retroviral RTs. The discovery, optimization and deployment of processive group II intron RTs provide us the opportunity to analyze RNA transcripts with single molecule resolution. MarathonRT, the most processive group II intron RT, has been extensively optimized for processive reverse transcription. In this chapter, we use MarathonRT to deliver a general protocol for long amplicon generation by RT-PCR, and also provide guidance for troubleshooting and further optimization.
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DNA Polimerase Dirigida por RNA , RNA , RNA/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase , ÍntronsRESUMO
BACKGROUND: This study aimed to identify factors associated with antibiotic-associated diarrhea (AAD) in patients in the department of intensive care medicine who received antibiotic monotherapy in order to reduce the incidence of AAD and improve rational use of antibiotics in these patients. AIM: To report the incidence of AAD and the factors associated with AAD in patients receiving antibiotic monotherapy. METHODS: The study used a single-center retrospective design. A total of 209 patients were enrolled. Patients were divided into two groups: No-AAD group (without AAD) and AAD group (with AAD). There were 45 cases in the AAD group and 164 cases in the no-AAD group. Clinical data of all patients were collected. Data were analyzed using SPSS (version 18.0), and statistical signiï¬cance was set at P < 0.05. RESULTS: The overall incidence of AAD was 21.53%. Age [odds ratio (OR) 1.022, 95% confidence interval (CI): 1.001-1.044, P = 0.040], proton pump inhibitor usage time (OR 1.129, 95%CI: 1.020-1.249, P = 0.019), antibiotic usage time (OR 1.163, 95%CI: 1.024-1.320, P = 0.020), and intensive care unit (ICU) stay time (OR 1.133, 95%CI: 1.041-1.234, P = 0.004) were associated with AAD in ICU patients receiving antibiotic monotherapy. mean ± SD ICU stay time was lower in the no-AAD group (8.49 ± 6.31 vs 15.89 ± 10.69, P < 0.001). However, there was no significant difference in ICU-related mortality rates between the two groups (P = 0.729). CONCLUSION: Older age, longer ICU stay time, duration of use of proton pump inhibitors, and duration of antibiotic increase the incidence of AAD in ICU patients receiving antibiotic monotherapy.
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Reverse transcriptase (RT) enzymes are indispensable tools for interrogating diverse aspects of RNA metabolism and transcriptome composition. Due to the growing interest in sequence and structural complexity of long RNA molecules, processive RT enzymes are now required for preserving linkage and information content in mixed populations of transcripts, and the low-processivity RT enzymes that are commercially available cannot meet this need. MarathonRT is encoded within a eubacterial group II intron, and it has been shown to efficiently copy highly structured long RNA molecules in a single pass. In this work, we systematically characterize MarathonRT as a tool enzyme and optimize its performance in a variety of applications that include single-cycle reverse transcription of long RNAs, dimethyl sulfate mutational profiling (DMS-MaP), selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), using ultra-long amplicons and the detection of natural RNA base modifications. By diversifying MarathonRT reaction protocols, we provide an upgraded suite of tools for cutting-edge RNA research and clinical application.
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Bactérias/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , RNA/química , RNA/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Conformação de Ácido Nucleico , RNA/genética , Análise de Sequência de RNARESUMO
For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.
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RNA de Transferência de Triptofano/metabolismo , Triptofano-tRNA Ligase/química , Triptofano/metabolismo , Valina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoleucina/química , Dados de Sequência Molecular , Mutação , Ligação Proteica , Bases de Schiff/química , Alinhamento de Sequência , Triptofano-tRNA Ligase/genética , Triptofano-tRNA Ligase/metabolismo , Valina/genéticaRESUMO
Bemisia tabaci has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we investigated whether hydroxyacid-oxoacid transhydrogenase (HOT) is involved in resistance to the neonicotinoid insecticide thiamethoxam in the whitefly. We cloned the full-length gene that encodes HOT in B. tabaci. Its cDNA contains a 1428-bp open reading frame encoding 475 amino acid residues. Then we evaluated the mRNA expression level of HOT in different developmental stages, and found HOT expression was significantly greater in thiamethoxam resistance adults than in thiamethoxam susceptible adults. Subsequently, seven field populations of B. tabaci adults were sampled, the expression of mRNA level of HOT significant positive correlated with thiamethoxam resistance level. At last, we used a modified gene silencing system to knock-down HOT expression in B. tabaci adults. The results showed that the HOT mRNA levels decreased by 57% and thiamethoxam resistance decreased significantly after 2 days of feeding on a diet containing HOT dsRNA. The results indicated that down-regulation of HOT expression decreases thiamethoxam resistance in B. tabaci adults.
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Oxirredutases do Álcool/genética , Hemípteros/enzimologia , Hemípteros/metabolismo , Proteínas de Insetos/genética , Inseticidas/toxicidade , Proteínas Mitocondriais/genética , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Oxazinas/toxicidade , Tiazóis/toxicidade , Oxirredutases do Álcool/metabolismo , Animais , Técnicas de Silenciamento de Genes , Hemípteros/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Proteínas Mitocondriais/metabolismo , Interferência de RNA , TiametoxamRESUMO
Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.
Assuntos
Códon , Escherichia coli , RNA de Transferência de Serina , Serina/metabolismo , Códon/genética , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA de Transferência de Serina/genética , RNA de Transferência de Serina/metabolismoRESUMO
OBJECTIVES: This meta-analysis summarized the risks that reintubation impose on ventilator-associated pneumonia (VAP) and mortality. BACKGROUND: Extubation failure increases the probability of poor clinical outcomes pertaining to mechanical ventilation. METHODS: Literature published during a 15-year period was retrieved from PubMed, Web of Knowledge databases, the Embase (Excerpa Medica database), and the Cochrane Library. Data involving reintubation, VAP, and mortality were extracted for a meta-analysis. RESULTS: Forty-one studies involving 29,923 patients were enrolled for the analysis. The summary odds ratio (OR) between VAP and reintubation was 7.57 (95% confidence interval [CI] = 3.63-15.81). The merged ORs for mortality in hospital and intensive care unit were 3.33 (95% CI = 2.02-5.49) and 7.50 (95% CI = 4.60-12.21), respectively. CONCLUSIONS: Reintubation can represent a threat to survival and increase the risk of VAP. The risk of mortality after reintubation differs between planned and unplanned extubation. Extubation failure is associated with a higher risk of VAP in the cardiac surgery population than in the general population.