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1.
Nucleic Acids Res ; 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34581811

RESUMO

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the ß-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.

2.
Nucleic Acids Res ; 49(15): 8757-8776, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379789

RESUMO

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.


Assuntos
Genes Bacterianos , Óperon , Biossíntese de Proteínas , RNA de Transferência/genética , Estresse Fisiológico/genética , Anabaena/genética , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Viabilidade Microbiana/genética , RNA de Transferência/metabolismo , Sequências Reguladoras de Ácido Nucleico
3.
mBio ; 12(3): e0113221, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34126764

RESUMO

Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence. IMPORTANCE Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate. It is thought that antibiotic bacterial persisters may cause reoccurring infections in the clinical setting. The molecular triggers and pathways that cause bacteria to enter into the persister state are not fully understood. Our results suggest that accumulation of deacylated tRNA is a trigger for antibiotic persistence independent of the RelA-dependent stringent response, a pathway thought to be required for persistence in many organisms. Overall, this provides a mechanism where changes in translation quality control in response to physiological cues can directly modulate bacterial persistence.

4.
Front Genet ; 11: 856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33014012

RESUMO

Bacterial oxidative stress responses are generally controlled by transcription factors that modulate the synthesis of RNAs with the aid of some sRNAs that control the stability, and in some cases the translation, of specific mRNAs. Here, we report that oxidative stress additionally leads to inactivation of tRNAGly in Escherichia coli, inducing a series of physiological changes. The observed inactivation of tRNAGly correlated with altered efficiency of translation of Gly codons, suggesting a possible mechanism of translational control of gene expression under oxidative stress. Changes in translation also depended on the availability of glycine, revealing a mechanism whereby bacteria modulate the response to oxidative stress according to the prevailing metabolic state of the cells.

5.
Genes (Basel) ; 11(10)2020 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-33081015

RESUMO

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

6.
Exp Cell Res ; 394(2): 112161, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32619498

RESUMO

Organisms encounter stress throughout their lives, and therefore require the ability to respond rapidly to environmental changes. Although transcriptional responses are crucial for controlling changes in gene expression, regulation at the translational level often allows for a faster response at the protein levels which permits immediate adaptation. The fidelity and robustness of protein synthesis are actively regulated under stress. For example, mistranslation can be beneficial to cells upon environmental changes and also alters cellular stress responses. Additionally, stress modulates both global and selective translational regulation through mechanisms including the change of aminoacyl-tRNA activity, tRNA pool reprogramming and ribosome heterogeneity. In this review, we draw on studies from both the prokaryotic and eukaryotic systems to discuss current findings of cellular adaptation at the level of translation, specifically translational fidelity and activity changes in response to a wide array of environmental stressors including oxidative stress, nutrient depletion, temperature variation, antibiotics and host colonization.


Assuntos
Adaptação Fisiológica , Biossíntese de Proteínas , Estresse Fisiológico , Animais , Humanos , Estresse Oxidativo , Ribossomos/metabolismo
7.
J Biol Chem ; 295(30): 10434-10445, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32518156

RESUMO

Bacteria must rapidly respond to both intracellular and environmental changes to survive. One critical mechanism to rapidly detect and adapt to changes in environmental conditions is control of gene expression at the level of protein synthesis. At each of the three major steps of translation-initiation, elongation, and termination-cells use stimuli to tune translation rate and cellular protein concentrations. For example, changes in nutrient concentrations in the cell can lead to translational responses involving mechanisms such as dynamic folding of riboswitches during translation initiation or the synthesis of alarmones, which drastically alter cell physiology. Moreover, the cell can fine-tune the levels of specific protein products using programmed ribosome pausing or inducing frameshifting. Recent studies have improved understanding and revealed greater complexity regarding long-standing paradigms describing key regulatory steps of translation such as start-site selection and the coupling of transcription and translation. In this review, we describe how bacteria regulate their gene expression at the three translational steps and discuss how translation is used to detect and respond to changes in the cellular environment. Finally, we appraise the costs and benefits of regulation at the translational level in bacteria.


Assuntos
Adaptação Fisiológica , Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Biossíntese de Proteínas/fisiologia
8.
RNA ; 26(8): 910-936, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32303649

RESUMO

The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.


Assuntos
Aminoacil-tRNA Sintetases/genética , RNA de Transferência/genética , Animais , Código Genético , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Aminoacilação de RNA de Transferência/genética
9.
PLoS Negl Trop Dis ; 14(2): e0007983, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32106219

RESUMO

The development of chemotherapies against eukaryotic pathogens is especially challenging because of both the evolutionary conservation of drug targets between host and parasite, and the evolution of strain-dependent drug resistance. There is a strong need for new nontoxic drugs with broad-spectrum activity against trypanosome parasites such as Leishmania and Trypanosoma. A relatively untested approach is to target macromolecular interactions in parasites rather than small molecular interactions, under the hypothesis that the features specifying macromolecular interactions diverge more rapidly through coevolution. We computed tRNA Class-Informative Features in humans and independently in eight distinct clades of trypanosomes, identifying parasite-specific informative features, including base pairs and base mis-pairs, that are broadly conserved over approximately 250 million years of trypanosome evolution. Validating these observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase (aaRS) interactions are a promising target for anti-trypanosomal drug discovery. From a marine natural products extract library, we identified several fractions with inhibitory activity toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the human homolog. These marine natural products extracts showed cross-reactivity towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our network predictions. We also identified Leishmania major threonyl-tRNA synthetase (ThrRS) inhibitors from the same library. We discuss why chemotherapies targeting multiple aaRSs should be less prone to the evolution of resistance than monotherapeutic or synergistic combination chemotherapies targeting only one aaRS.


Assuntos
Alanina-tRNA Ligase/antagonistas & inibidores , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Treonina-tRNA Ligase/antagonistas & inibidores , Trypanosoma/efeitos dos fármacos , Alanina-tRNA Ligase/genética , Alanina-tRNA Ligase/metabolismo , Antiprotozoários/química , Inibidores Enzimáticos/química , Humanos , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmaniose/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Trypanosoma/enzimologia , Trypanosoma/genética , Tripanossomíase/parasitologia
10.
J Biol Chem ; 295(5): 1402-1410, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31862734

RESUMO

ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.


Assuntos
Alanina-tRNA Ligase/metabolismo , Diamino Aminoácidos/metabolismo , Aminoacilação de RNA de Transferência , Alanina/química , Alanina/metabolismo , Diamino Aminoácidos/química , Cromatografia Líquida , Expressão Gênica , Humanos , Cinética , Espectrometria de Massas , Serina/química , Serina/metabolismo , Serina-tRNA Ligase/metabolismo
11.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848288

RESUMO

Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated. Despite that interpretation, there is evidence that some aminoacylation errors may be regulated, and thus provide a benefit to the cell, while others are clearly detrimental. Here, we show that while it has been suggested that regulated Thr-to-Ser substitutions may be beneficial, there is a threshold beyond which these errors are detrimental. In contrast, we show that errors mediated by alanyl-tRNA synthetase (AlaRS) are not well tolerated and induce a global stress response that leads to gross perturbation of the Escherichia coli proteome, with potentially catastrophic effects on fitness and viability. Tolerance for Ala mistranslation appears to be much lower than with other translational errors, consistent with previous reports of multiple proofreading mechanisms targeting mischarged tRNAAla These results demonstrate the essential role of aminoacyl-tRNA proofreading in optimizing cellular fitness and suggest that any potentially beneficial effects of mistranslation may be confined to specific amino acid substitutions.IMPORTANCE Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteoma , Proteômica , Membrana Celular/metabolismo , Biossíntese de Proteínas , Proteômica/métodos , RNA de Transferência de Serina/química , RNA de Transferência de Serina/genética , Especificidade por Substrato , Aminoacilação de RNA de Transferência
12.
PLoS Pathog ; 15(9): e1007948, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31560731

RESUMO

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.


Assuntos
Salmonella typhimurium/fisiologia , Salmonella typhimurium/patogenicidade , Animais , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Genes Bacterianos , Aptidão Genética , Macrófagos/microbiologia , Camundongos , Plasmídeos/genética , Células RAW 264.7 , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Virulência/genética , Virulência/fisiologia
13.
Open Biol ; 9(7): 190051, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31288624

RESUMO

Many antibiotics available in the clinic today directly inhibit bacterial translation. Despite the past success of such drugs, their efficacy is diminishing with the spread of antibiotic resistance. Through the use of ribosomal modifications, ribosomal protection proteins, translation elongation factors and mistranslation, many pathogens are able to establish resistance to common therapeutics. However, current efforts in drug discovery are focused on overcoming these obstacles through the modification or discovery of new treatment options. Here, we provide an overview for common mechanisms of resistance to translation-targeting drugs and summarize several important breakthroughs in recent drug development.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Biossíntese de Proteínas/fisiologia , Animais , Descoberta de Drogas , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Humanos , Terapia de Alvo Molecular/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
14.
IUBMB Life ; 71(8): 1141-1149, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31241862

RESUMO

Mutations in nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl-tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild-type enzyme with reduced solubility in vitro. The presence of aggregation-prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function. © 2019 IUBMB Life, 71(8): 1141-1149, 2019 © 2019 IUBMB Life, 71(8):1141-1149, 2019.


Assuntos
Esclerose Cerebral Difusa de Schilder/enzimologia , Mitocôndrias/enzimologia , Paraplegia/enzimologia , Fenilalanina-tRNA Ligase/fisiologia , Trifosfato de Adenosina/química , Aminoacilação , Esclerose Cerebral Difusa de Schilder/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Luz , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Mutação , Paraplegia/genética , Tamanho da Partícula , Fenilalanina/química , Fenilalanina-tRNA Ligase/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Solubilidade , Temperatura
15.
IUBMB Life ; 71(8): 1150-1157, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31135095

RESUMO

Translation is the most error-prone process in protein synthesis; however, it is important that accuracy is maintained because erroneous translation has been shown to affect all domains of life. Translational quality control is maintained by both proteins and RNA through intricate processes. The aminoacyl-tRNA synthetases help maintain high levels of translational accuracy through the esterification of tRNA and proofreading mechanisms. tRNA is often recognized by an aminoacyl-tRNA synthetase in a sequence and structurally dependent manner, sometimes involving modified nucleotides. Additionally, some proofreading mechanisms of aminoacyl-tRNA synthetases require tRNA elements for hydrolysis of a noncognate aminoacyl-tRNA. Finally, tRNA is also important for proper decoding of the mRNA message by codon and anticodon pairing. Here, recent developments regarding the importance of tRNA in maintenance of translational accuracy are reviewed. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1150-1157, 2019.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , RNA de Transferência/genética , Animais , Anticódon , Códon , Escherichia coli/enzimologia , Ésteres , Humanos , Camundongos , Nucleotídeos/genética , Organelas/metabolismo , Estresse Oxidativo , Fenótipo , RNA Mensageiro/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/enzimologia
16.
Proc Natl Acad Sci U S A ; 116(20): 10058-10063, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31036643

RESUMO

Accurate translation of the genetic code is maintained in part by aminoacyl-tRNA synthetases (aaRS) proofreading mechanisms that ensure correct attachment of a cognate amino acid to a transfer RNA (tRNA). During environmental stress, such as oxidative stress, demands on aaRS proofreading are altered by changes in the availability of cytoplasmic amino acids. For example, oxidative stress increases levels of cytotoxic tyrosine isomers, noncognate amino acids normally excluded from translation by the proofreading activity of phenylalanyl-tRNA synthetase (PheRS). Here we show that oxidation of PheRS induces a conformational change, generating a partially unstructured protein. This conformational change does not affect Phe or Tyr activation or the aminoacylation activity of PheRS. However, in vitro and ex vivo analyses reveal that proofreading activity to hydrolyze Tyr-tRNAPhe is increased during oxidative stress, while the cognate Phe-tRNAPhe aminoacylation activity is unchanged. In HPX-, Escherichia coli that lack reactive oxygen-scavenging enzymes and accumulate intracellular H2O2, we found that PheRS proofreading is increased by 11%, thereby providing potential protection against hazardous cytoplasmic m-Tyr accumulation. These findings show that in response to oxidative stress, PheRS proofreading is positively regulated without negative effects on the enzyme's housekeeping activity in translation. Our findings also illustrate that while the loss of quality control and mistranslation may be beneficial under some conditions, increased proofreading provides a mechanism for the cell to appropriately respond to environmental changes during oxidative stress.


Assuntos
Fenilalanina-tRNA Ligase/metabolismo , Biossíntese de Proteínas , Escherichia coli , Oxirredução , Estresse Oxidativo , Conformação Proteica , Salmonella enterica
17.
Proc Natl Acad Sci U S A ; 115(43): 11072-11077, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30297417

RESUMO

Elongation factor P (EF-P) is a universally conserved translation factor that alleviates ribosome pausing at polyproline (PPX) motifs by facilitating peptide bond formation. In the absence of EF-P, PPX peptide bond formation can limit translation rate, leading to pleotropic phenotypes including slowed growth, increased antibiotic sensitivity, and loss of virulence. In this study, we observe that many of these phenotypes are dependent on growth rate. Limiting growth rate suppresses a variety of detrimental phenotypes associated with ribosome pausing at PPX motifs in the absence of EF-P. Polysome levels are also similar to wild-type under slow growth conditions, consistent with global changes in ribosome queuing in cells without EF-P when growth rate is decreased. Inversely, under high protein synthesis demands, we observe that Escherichia coli lacking EF-P have reduced fitness. Our data demonstrate that EF-P-mediated relief of ribosome queuing is required to maintain proteome homeostasis under conditions of high translational demands.


Assuntos
Escherichia coli/metabolismo , Escherichia coli/fisiologia , Homeostase/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , Proteoma/metabolismo , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Elongação Traducional da Cadeia Peptídica/fisiologia , Peptídeos/metabolismo , Fenótipo , Polirribossomos/metabolismo , Ribossomos/metabolismo , Virulência/fisiologia
18.
Ann Clin Transl Neurol ; 5(9): 1128-1133, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30250868

RESUMO

Mutations in FARS2, the gene encoding the mitochondrial phenylalanine-tRNA synthetase (mtPheRS), have been linked to a range of phenotypes including epileptic encephalopathy, developmental delay, and motor dysfunction. We report a 9-year-old boy with novel compound heterozygous variants of FARS2, presenting with a pure spastic paraplegia syndrome associated with bilateral signal abnormalities in the dentate nuclei. Exome sequencing identified a paternal nonsense variant (Q216X) lacking the catalytic core and anticodon-binding regions, and a maternal missense variant (P136H) possessing partial enzymatic activity. This case confirms and expands the phenotype related to FARS2 mutations with regards to clinical presentation and neuroimaging findings.

19.
Biochem Biophys Res Commun ; 502(4): 450-455, 2018 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-29859934

RESUMO

It is widely believed that if a high number of genes are found for any tRNA in a rapidly replicating bacteria, then the cytoplasmic levels of that tRNA will be high and an open reading frame containing a higher frequency of the complementary codon will be translated faster. This idea is based on correlations between the number of tRNA genes, tRNA concentration and the frequency of codon usage observed in a limited number of strains as well as from the fact that artificially changing the number of tRNA genes alters translation efficiency and consequently the amount of properly folded protein synthesized. tRNA gene number may greatly vary in a genome due to duplications, deletions and lateral transfer which in turn would alter the levels and functionality of many proteins. Such changes are potentially deleterious for fitness and as a result it is expected that changes in tRNA gene numbers should be accompanied by a modification of the frequency of codon usage. In contrast to this model, when comparing the number of tRNA genes and the frequency of codon usage of several Salmonella enterica and Escherichia coli strains we found that changes in the number of tRNA genes are not correlated to changes in codon usage. Furthermore, these changes are not correlated with a change in the efficiency of codon translation. These results suggest that once a genome gains or loses tRNA genes, it responds by modulating the concentrations of tRNAs rather than modifying its frequency of codon usage.


Assuntos
Códon/genética , Enterobacteriaceae/genética , Genes Bacterianos , Escherichia coli/genética , Dosagem de Genes , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA de Transferência/genética , Salmonella enterica/genética
20.
Biochim Biophys Acta Gen Subj ; 1862(8): 1801-1809, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29723545

RESUMO

BACKGROUND: Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis. METHODS: Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity. RESULTS: ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress. CONCLUSION: Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly. GENERAL SIGNIFICANCE: The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.


Assuntos
Mitocôndrias/enzimologia , Estresse Oxidativo , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato
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