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1.
Nat Commun ; 11(1): 4531, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913225

RESUMO

RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5'-to-3' direction, at ~60 nt s-1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.


Assuntos
Fusobacterium/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA/genética , RNA Bacteriano/genética , Riboswitch/genética , Aminoimidazol Carboxamida/metabolismo , Fusobacterium/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Ribonucleotídeos/metabolismo , Imagem Individual de Molécula , Transcrição Genética
2.
PLoS One ; 15(8): e0237127, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756602

RESUMO

BACKGROUND: The global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown. METHODS: We compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay. RESULTS: Of the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%). CONCLUSION: We found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Staphylococcus aureus Resistente à Meticilina/genética , Pneumonia Viral/diagnóstico , Manejo de Espécimes/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Nasofaringe/microbiologia , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Bacteriano/análise , RNA Bacteriano/metabolismo , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
3.
Nature ; 585(7823): 124-128, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848247

RESUMO

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Transcrição Genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Filogenia , RNA Bacteriano/biossíntese , RNA Bacteriano/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Ribossomos/metabolismo , Riboswitch/genética
4.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
5.
Nat Commun ; 11(1): 3363, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620750

RESUMO

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.


Assuntos
Regulação Bacteriana da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Doenças Negligenciadas/imunologia , Orientia tsutsugamushi/genética , Tifo por Ácaros/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Genoma Bacteriano , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Sequências Repetitivas Dispersas/genética , Camundongos , Doenças Negligenciadas/microbiologia , Orientia tsutsugamushi/imunologia , Orientia tsutsugamushi/patogenicidade , Proteômica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA-Seq , Tifo por Ácaros/microbiologia , Transcrição Genética , Sequenciamento Completo do Exoma
6.
Phys Rev Lett ; 125(2): 028103, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32701325

RESUMO

Bacterial ribosomes are composed of one-third protein and two-thirds RNA by mass. The predominance of RNA is often attributed to a primordial RNA world, but why exactly two-thirds remains a long-standing mystery. Here we present a quantitative analysis, based on the kinetics of ribosome self-replication, demonstrating that the 1∶2 protein-to-RNA mass ratio uniquely maximizes cellular growth rates in E. coli. A previously unrecognized growth law, and an invariant of bacterial growth, also follow from our analysis. The growth law reveals that the ratio between the number of ribosomes and the number of polymerases making ribosomal RNA is proportional to the cellular doubling time. The invariant is conserved across growth conditions and specifies how key microscopic parameters in the cell, such as transcription and translation rates, are coupled to cellular physiology. Quantitative predictions from the growth law and invariant are shown to be in excellent agreement with E. coli data despite having no fitting parameters. Our analysis can be readily extended to other bacteria once data become available.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Modelos Biológicos , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Escherichia coli/genética , Ribossomos/genética
7.
Proc Natl Acad Sci U S A ; 117(28): 16333-16338, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601241

RESUMO

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.


Assuntos
Biossíntese de Proteínas/genética , RNA de Transferência/química , RNA de Transferência/genética , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Pareamento de Bases , Códon/genética , Códon/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
8.
RNA ; 26(10): 1448-1463, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32646969

RESUMO

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Poli A/metabolismo , Domínios Proteicos/fisiologia , RNA Bacteriano/metabolismo , Proteínas Repressoras/metabolismo , Salmonella enterica/metabolismo
9.
PLoS One ; 15(7): e0235633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628709

RESUMO

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Doxiciclina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Cadeia Dupla/metabolismo
10.
Nat Commun ; 11(1): 2823, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499480

RESUMO

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Neisseria meningitidis/genética , Conformação de Ácido Nucleico , Estresse Oxidativo , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
11.
Nat Commun ; 11(1): 2837, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503992

RESUMO

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5'-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.


Assuntos
Íntrons/genética , Precursores de RNA/metabolismo , Processamento de RNA , RNA Bacteriano/metabolismo , Spliceossomos/metabolismo , Bacillaceae/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutagênese , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA Bacteriano/genética , Análise Espaço-Temporal
12.
Nucleic Acids Res ; 48(12): 6970-6979, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479610

RESUMO

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.


Assuntos
Aptâmeros de Nucleotídeos/genética , Conformação de Ácido Nucleico , Ribonucleotídeos/genética , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Firmicutes/genética , Firmicutes/ultraestrutura , Ligantes , Purinas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Ribonucleotídeos/química
13.
Nucleic Acids Res ; 48(14): e81, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32504488

RESUMO

RNA secondary structure around translation initiation sites strongly affects the abundance of expressed proteins in Escherichia coli. However, detailed secondary structural features governing protein abundance remain elusive. Recent advances in high-throughput DNA synthesis and experimental systems enable us to obtain large amounts of data. Here, we evaluated six types of structural features using two large-scale datasets. We found that accessibility, which is the probability that a given region around the start codon has no base-paired nucleotides, showed the highest correlation with protein abundance in both datasets. Accessibility showed a significantly higher correlation (Spearman's ρ = 0.709) than the widely used minimum free energy (0.554) in one of the datasets. Interestingly, accessibility showed the highest correlation only when it was calculated by a log-linear model, indicating that the RNA structural model and how to utilize it are important. Furthermore, by combining the accessibility and activity of the Shine-Dalgarno sequence, we devised a method for predicting protein abundance more accurately than existing methods. We inferred that the log-linear model has a broader probabilistic distribution than the widely used Turner energy model, which contributed to more accurate quantification of ribosome accessibility to translation initiation sites.


Assuntos
Escherichia coli/genética , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 5' não Traduzidas , Algoritmos , Códon de Iniciação/metabolismo , Conjuntos de Dados como Assunto , Escherichia coli/metabolismo , Previsões , Modelos Lineares , Aprendizado de Máquina , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/química , RNA Mensageiro/química , Sequências Reguladoras de Ácido Ribonucleico , Relação Estrutura-Atividade
14.
Mol Cell ; 79(2): 280-292.e8, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32533919

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking ten TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems do not strongly promote survival following individual stresses.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Sistemas Toxina-Antitoxina , Transcrição Genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Proteólise , RNA Bacteriano/metabolismo , RNA-Seq , Sistemas Toxina-Antitoxina/genética
15.
PLoS Genet ; 16(6): e1008837, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584816

RESUMO

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Perfilação da Expressão Gênica , Peptídeo Sintases/genética , Peptídeo Sintases/isolamento & purificação , Peptídeo Sintases/metabolismo , Biossíntese de Proteínas , Proteoma/genética , Proteômica , Pseudomonas fluorescens/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rizosfera , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/genética
16.
Mol Biotechnol ; 62(6-7): 326-334, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32227298

RESUMO

The study of translation initiation in prokaryotes assumes that there should be a mechanism different from the canonical model, which postulates the formation of the pre-initiation complex through the interaction of the Shine-Dalgarno sequence (SD) at the 5'-end of mRNA and the anti-Shine-Dalgarno site at the 3'-end of 16S rRNA. In this paper we've studied the effect of TPS (Translation-initiation Promoting Site) on ß-glucuronidase expression in E. coli cells at different cultivation temperatures. The examined leader sequences were cloned into the pET23c plasmid upstream the ß-glucuronidase gene; protein expression was performed in E. coli BL21 (DE3) cells. ß-glucuronidase activity was measured in bacterial cell extracts via paranitrophenyl b-D-glucuronide assay. The quantity of expressed protein was measured by Western blotting with following densitometry. It was shown that TPS increases the level of protein expression at stressful conditions (10 °C and 44 °C) 5-8 times compared to control. The combination of TPS and SD sites in the 5'-leader sequence of the mRNA created an enhancer that increased the expression level 2-3.6 times compared to a single SD-sequence. Based on the obtained data and the computer modeling of interaction between 16S rRNA and TPS, we proposed an alternative variation of prokaryotic translation initiation.


Assuntos
Escherichia coli/metabolismo , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/fisiologia , Escherichia coli/genética , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo
17.
Nucleic Acids Res ; 48(8): 4507-4520, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32170306

RESUMO

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Escherichia coli/genética , Técnicas Genéticas , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
18.
PLoS One ; 15(2): e0228199, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040520

RESUMO

In the present study, we identified salt stress tolerant genes from the marine bacterium Staphylococcus sp. strain P-TSB-70 through transcriptome sequencing. In favour of whole-genome transcriptome profiling of Staphylococcus sp. strain P-TSB-70 (GenBank Accn. No. KP117091) which tolerated upto 20% NaCl stress, the strain was cultured in the laboratory condition with 20% NaCl stress. Transcriptome analyses were performed by SOLiD4.0 sequencing technology from which 10280 and 9612 transcripts for control and treated, respectively, were obtained. The coverage per base (CPB) statistics were analyzed for both the samples. Gene ontology (GO) analysis has been categorized at varied graph levels based on three primary ontology studies viz. cellular components, biological processes, and molecular functions. The KEGG analysis of the assembled transcripts using KAAS showed presumed components of metabolic pathways which perhaps implicated in diverse metabolic pathways responsible for salt tolerance viz. glycolysis/gluconeogenesis, oxidative phosphorylation, glutathione metabolism, etc. further involving in salt tolerance. Overall, 90 salt stress tolerant genes were identified as of 186 salt-related transcripts. Several genes have been found executing normally in the TCA cycle pathway, integral membrane proteins, generation of the osmoprotectants, enzymatic pathway associated with salt tolerance. Recognized genes fit diverse groups of salt stress genes viz. abc transporter, betaine, sodium antiporter, sodium symporter, trehalose, ectoine, and choline, that belong to different families of genes involved in the pathway of salt stress. The control sample of the bacterium showed elevated high proportion of transcript contigs (29%) while upto 20% salt stress treated sample of the bacterium showed a higher percentage of transcript contigs (31.28%). A total of 1,288 and 1,133 transcript contigs were measured entirely as novel transcript contigs in both control and treated samples, respectively. The structure and function of 10 significant salt stress tolerant genes of Staphylococcus sp. have been analyzed in this study. The information acquired in the present study possibly used to recognize and clone the salt stress tolerant genes and support in developing the salt stress-tolerant plant varieties to expand the agricultural productivity in the saline system.


Assuntos
Tolerância ao Sal/genética , Água do Mar/microbiologia , Staphylococcus/genética , Transcriptoma , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mapeamento de Sequências Contíguas , Perfilação da Expressão Gênica , Genoma Bacteriano , Índia , Redes e Vias Metabólicas/genética , Estrutura Terciária de Proteína , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Staphylococcus/isolamento & purificação
19.
PLoS Comput Biol ; 16(1): e1007643, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004314

RESUMO

For a genetically identical microbial population, multi-gene expression in various environments requires effective allocation of limited resources and precise control of heterogeneity among individual cells. However, it is unclear how resource allocation and cell-to-cell variation jointly shape the overall performance. Here we demonstrate a Simpson's paradox during overexpression of multiple genes: two competing proteins in single cells correlated positively for every induction condition, but the overall correlation was negative. Yet this phenomenon was not observed between two competing mRNAs in single cells. Our analytical framework shows that the phenomenon arises from competition for translational resource, with the correlation modulated by both mRNA and ribosome variability. Thus, heterogeneity plays a key role in single-cell multi-gene expression and provides the population with an evolutionary advantage, as demonstrated in this study.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Expressão Gênica/genética , Genes Bacterianos/genética , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fenômenos Genéticos/genética , Modelos Estatísticos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Biochim Biophys Acta Gene Regul Mech ; 1863(3): 194506, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32068131

RESUMO

Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.


Assuntos
RNA Bacteriano/química , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico
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