RESUMO
Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.
Assuntos
Reparo do DNA , RNA Polimerases Dirigidas por DNA , Escherichia coli , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Ribonucleotídeos/metabolismoRESUMO
RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome-wide distribution of more persistent backtracking are unknown. Consequently, we developed a method to directly sequence the extruded, "backtracked" 3' RNA. Our data show that RNA Pol II slides backward more than 20 nt in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where RNA Pol II pauses near promoters and intron-exon junctions and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking can potentially affect diverse gene expression programs.
Assuntos
RNA Polimerase II , RNA , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA/genética , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/genéticaRESUMO
Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Fator sigma/metabolismo , Terminação da Transcrição Genética , Regiões 5' não TraduzidasRESUMO
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.
Assuntos
Antibacterianos , Sítios de Ligação , RNA Polimerases Dirigidas por DNA , Escherichia coli , Mutação , Rifampina , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Quebras de DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Nucleotídeos/deficiência , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas , Rifampina/química , Rifampina/metabolismo , Rifampina/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Nitric oxide (NO) is an important signaling molecule in multicellular organisms. Most animals produce NO from L-arginine via a family of dedicated enzymes known as NO synthases (NOSes). A rare exception is the roundworm Caenorhabditis elegans, which lacks its own NOS. However, in its natural environment, C. elegans feeds on Bacilli that possess functional NOS. Here, we demonstrate that bacterially derived NO enhances C. elegans longevity and stress resistance via a defined group of genes that function under the dual control of HSF-1 and DAF-16 transcription factors. Our work provides an example of interspecies signaling by a small molecule and illustrates the lifelong value of commensal bacteria to their host.
Assuntos
Bacillus subtilis , Caenorhabditis elegans/fisiologia , Longevidade , Óxido Nítrico/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Dieta , Fatores de Transcrição Forkhead , Trato Gastrointestinal/microbiologia , Temperatura , Fatores de Transcrição/metabolismoRESUMO
In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcriptionreplication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.
Assuntos
Escherichia coli/genética , Evolução Molecular , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese/genética , Taxa de Mutação , Antibacterianos/farmacologia , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Fluoroquinolonas/farmacologia , Loci Gênicos/efeitos dos fármacos , Loci Gênicos/genética , Variação Genética/efeitos dos fármacos , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Mutação INDEL/genética , Mutagênese/efeitos dos fármacos , Nucleotídeos/genética , Nucleotídeos/metabolismo , Estresse Oxidativo/genética , Transcrição Gênica/genéticaRESUMO
Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.
Assuntos
Proteínas de Transporte/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Terminação da Transcrição Genética , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Linhagem Celular , Técnicas de Inativação de Genes , Histonas/metabolismo , Camundongos , Mioblastos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Ubiquitinação/genéticaRESUMO
RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , RNA Antissenso/metabolismo , Transcrição Gênica , Regulação Bacteriana da Expressão GênicaRESUMO
In this issue of Molecular Cell, Mariner et al. (2008) demonstrate that Alu RNA from a human SINE represses RNA polymerase II transcription during heat shock. This noncoding RNA is the first example of a "protein-like" transcription factor with a distinct modular architecture.
Assuntos
RNA não Traduzido , Elementos Nucleotídeos Curtos e Dispersos/genética , Transcrição Gênica , Animais , Resposta ao Choque Térmico/fisiologia , Humanos , Conformação de Ácido Nucleico , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of ß and σ70 subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis.
Assuntos
Proteínas de Escherichia coli , Fatores Genéricos de Transcrição , Escherichia coli/metabolismo , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Bactérias/metabolismoRESUMO
Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.
RESUMO
RNA polymerase II (pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome wide distribution of more persistent backtracking is unknown. Consequently, we developed a novel method to directly sequence the extruded, "backtracked" 3' RNA. Our data shows that pol II slides backwards more than 20 nucleotides in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where pol II pauses near promoters and intron-exon junctions, and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking has the potential to affect diverse gene expression programs.
RESUMO
The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation.
Assuntos
Resposta ao Choque Térmico/fisiologia , RNA não Traduzido/fisiologia , Animais , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Escherichia coli , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Glutathione (GSH) is the most abundant cellular antioxidant. As reactive oxygen species (ROS) are widely believed to promote aging and age-related diseases, and antioxidants can neutralize ROS, it follows that GSH and its precursor, N-acetyl cysteine (NAC), are among the most popular dietary supplements. However, the long- term effects of GSH or NAC on healthy animals have not been thoroughly investigated. We employed C. elegans to demonstrate that chronic administration of GSH or NAC to young or aged animals perturbs global gene expression, inhibits skn-1-mediated transcription, and accelerates aging. In contrast, limiting the consumption of dietary thiols, including those naturally derived from the microbiota, extended lifespan. Pharmacological GSH restriction activates the unfolded protein response and increases proteotoxic stress resistance in worms and human cells. It is thus advantageous for healthy individuals to avoid excessive dietary antioxidants and, instead, rely on intrinsic GSH biosynthesis, which is fine-tuned to match the cellular redox status and to promote homeostatic ROS signaling.
Assuntos
Acetilcisteína/farmacologia , Envelhecimento/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Glutationa/farmacologia , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Suplementos Nutricionais , Escherichia coli , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Masculino , Paraquat/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.
Assuntos
Antibacterianos/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Biofilmes , Cristalografia por Raios X , Cistationina gama-Liase/química , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Descoberta de Drogas , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Tolerância a Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source of targets for combination antimicrobial therapies. Using transposon sequencing, we carried out a genome-wide analysis of fitness cost in a clinically common rpoB H526Y mutant. We find that genes whose products enable increased transcription elongation rates compound the fitness costs of resistance whereas genes whose products function in cell wall synthesis and division mitigate it. We validate our findings by showing that the cell wall synthesis and division defects of rpoB H526Y result from an increased transcription elongation rate that is further exacerbated by the activity of the uracil salvage pathway and unresponsiveness of the mutant RNAP to the alarmone ppGpp. We applied our findings to identify drugs that inhibit more readily rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic-resistant mutants should expedite the discovery of new combination therapies and delineate cellular pathways that underlie the molecular mechanisms of cost.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Rifampina/farmacologia , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana , Genoma Bacteriano , Mutação , Transcrição GênicaRESUMO
The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bgamma, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bgamma. We also show that infection by L. pneumophila leads to eEF1A-mediated activation of the heat shock regulatory protein HSF1 in a virulence-dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila-infected cells.
Assuntos
Proteínas de Transporte/fisiologia , Fenômenos Fisiológicos Celulares , Interações Hospedeiro-Patógeno , Legionella pneumophila/patogenicidade , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Biossíntese de Proteínas , Fatores de Virulência/fisiologia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição de Choque Térmico , Humanos , Camundongos , Mutação de Sentido Incorreto , Ligação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse Fisiológico , Fatores de Transcrição/biossíntese , Fatores de Virulência/genéticaRESUMO
The heat shock (HS) response is the major cellular defense mechanism against acute exposure to environmental stresses. The hallmark of the HS response, which is conserved in all eukaryotes, is the rapid and massive induction of expression of a set of cytoprotective genes. Most of the induction occurs at the level of transcription. The master regulator, heat shock transcription factor (HSF, or HSF1 in vertebrates), is responsible for the induction of HS gene transcription in response to elevated temperature. Under normal conditions HSF is present in the cell as an inactive monomer. During HS, HSF trimerizes and binds to a consensus sequence in the promoter of HS genes, stimulating their transcription by up to 200-fold. We have shown that a large, noncoding RNA, HSR1, and the translation elongation factor eEF1A form a complex with HSF during HS and are required for its activation.
Assuntos
Resposta ao Choque Térmico/genética , Biologia Molecular/métodos , RNA não Traduzido/isolamento & purificação , Células 3T3 , Animais , Extratos Celulares , Proteínas de Ligação a DNA/genética , Fator de Iniciação 1 em Eucariotos/isolamento & purificação , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Camundongos , RNA não Traduzido/genética , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H2S). Bacterial H2S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H2S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H2S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H2S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H2S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sulfeto de Hidrogênio/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Mapeamento Cromossômico , DNA Bacteriano , Proteínas de Ligação a DNA/genética , Dissacarídeos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Ligação Proteica , RNA Mensageiro , Regulon , Fatores de Transcrição/genéticaRESUMO
Large noncoding RNAs (lncRNAs) have emerged as key players in regulating various fundamental cellular processes. Recent reports identify a functional lncRNA, Evf-2, that operates during development to control the expression of specific homeodomain proteins, and they provide important insights into the mechanism of cooperation between a newly discovered nuclear receptor co-repressor protein (SLIRP) and steroid receptor activator RNA. Evf-2 is the first example of lncRNA directly involved in organogenesis in vertebrates.