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1.
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
2.
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
3.
BMC Bioinformatics ; 21(1): 15, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931703

RESUMO

BACKGROUND: Seed and accessibility constraints are core features to enable highly accurate sRNA target screens based on RNA-RNA interaction prediction. Currently, available tools provide different (sets of) constraints and default parameter sets. Thus, it is hard to impossible for users to estimate the influence of individual restrictions on the prediction results. RESULTS: Here, we present a systematic assessment of the impact of established and new constraints on sRNA target prediction both on a qualitative as well as computational level. This is done exemplarily based on the performance of IntaRNA, one of the most exact sRNA target prediction tools. IntaRNA provides various ways to constrain considered seed interactions, e.g. based on seed length, its accessibility, minimal unpaired probabilities, or energy thresholds, beside analogous constraints for the overall interaction. Thus, our results reveal the impact of individual constraints and their combinations. CONCLUSIONS: This provides both a guide for users what is important and recommendations for existing and upcoming sRNA target prediction approaches.We show on a large sRNA target screen benchmark data set that only by altering the parameter set, IntaRNA recovers 30% more verified interactions while becoming 5-times faster. This exemplifies the potential of seed, accessibility and interaction constraints for sRNA target prediction.


Assuntos
Bactérias/genética , Biologia Computacional/métodos , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/química , Bactérias/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo
4.
Nucleic Acids Res ; 48(4): 2144-2155, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31965171

RESUMO

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.


Assuntos
Carbono-Nitrogênio Ligases/química , RNA Polimerases Dirigidas por DNA/química , RNA Bacteriano/química , Transcrição Genética , Bacillus subtilis/química , Bacillus subtilis/genética , Carbono-Nitrogênio Ligases/genética , Cristalografia por Raios X , DNA/química , DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Fator sigma/química , Fator sigma/genética , Uridina Trifosfato/química , Uridina Trifosfato/genética
5.
PLoS Pathog ; 16(1): e1008184, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951643

RESUMO

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.


Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/metabolismo , Regiões 5' não Traduzidas , Animais , Toxinas Bacterianas/química , Feminino , Humanos , Sequências Repetidas Invertidas , Camundongos , Camundongos Endogâmicos BALB C , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Temperatura , Virulência , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidade
6.
J Chem Theory Comput ; 16(1): 688-699, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31751512

RESUMO

Argonaute (Ago) protein plays a central role in silencing gene expression by binding a "guide" strand to the base-pair with a complementary mRNA and degrading the mRNA. The current understanding of how Ago-guide and Ago-guide-mRNA complexes assemble is based mainly on static crystal structures; the associated kinetic pathways remain unknown/unclear. By simulating the successive binding of guide/target strand to Thermus thermophilus Ago (TtAgo) and computing the respective free energy landscapes, we directly visualize how TtAgo silencing complexes form and function. We show that the guide binding rate depends on its initial loading position onto TtAgo. Subsequent target recognition beyond the scissile 10-11 nucleotides must overcome a substantial energy barrier for TtAgo's nucleotide-binding groove to expand widely. This work reveals novel roles for the core TtAgo domains and shows how the kinetic barriers that must be overcome for critical structural changes to occur lead to target repression/cleavage.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , RNA Mensageiro/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonauta/química , Proteínas de Bactérias/química , Inativação Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , Termodinâmica , Thermus thermophilus/química , Thermus thermophilus/genética
7.
Nature ; 577(7789): 271-274, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31853065

RESUMO

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1-3. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.


Assuntos
Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , DNA Bacteriano/química , Vibrio cholerae/química , Microscopia Crioeletrônica , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica , Estrutura Quaternária de Proteína , RNA Bacteriano/química , Vibrio cholerae/genética
8.
PLoS One ; 14(12): e0225475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790434

RESUMO

Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Técnicas Bacteriológicas/métodos , Fragmentação do DNA/efeitos dos fármacos , Detergentes/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , DNA Bacteriano/química , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/efeitos da radiação , Hidrólise/efeitos da radiação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/efeitos da radiação , Micro-Ondas , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Oxigênio/análise , Oxigênio/metabolismo , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , Estabilidade de RNA/efeitos da radiação , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , RNA Bacteriano/efeitos da radiação , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/efeitos da radiação , Temperatura , Fatores de Tempo , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/efeitos da radiação
9.
PLoS Genet ; 15(12): e1008456, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830036

RESUMO

How genomes are organized within cells and how the 3D architecture of a genome influences cellular functions are significant questions in biology. A bacterial genomic DNA resides inside cells in a highly condensed and functionally organized form called nucleoid (nucleus-like structure without a nuclear membrane). The Escherichia coli chromosome or nucleoid is composed of the genomic DNA, RNA, and protein. The nucleoid forms by condensation and functional arrangement of a single chromosomal DNA with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. Although a high-resolution structure of a bacterial nucleoid is yet to come, five decades of research has established the following salient features of the E. coli nucleoid elaborated below: 1) The chromosomal DNA is on the average a negatively supercoiled molecule that is folded as plectonemic loops, which are confined into many independent topological domains due to supercoiling diffusion barriers; 2) The loops spatially organize into megabase size regions called macrodomains, which are defined by more frequent physical interactions among DNA sites within the same macrodomain than between different macrodomains; 3) The condensed and spatially organized DNA takes the form of a helical ellipsoid radially confined in the cell; and 4) The DNA in the chromosome appears to have a condition-dependent 3-D structure that is linked to gene expression so that the nucleoid architecture and gene transcription are tightly interdependent, influencing each other reciprocally. Current advents of high-resolution microscopy, single-molecule analysis and molecular structure determination of the components are expected to reveal the total structure and function of the bacterial nucleoid.


Assuntos
DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Escherichia coli/crescimento & desenvolvimento , RNA Bacteriano/química , DNA Super-Helicoidal/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Molecular , Imagem Individual de Molécula
11.
Nat Struct Mol Biol ; 26(12): 1114-1122, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792448

RESUMO

T-box riboswitches are modular bacterial noncoding RNAs that sense and regulate amino acid availability through direct interactions with tRNAs. Between the 5' anticodon-binding stem I domain and the 3' amino acid sensing domains of most T-boxes lies the stem II domain of unknown structure and function. Here, we report a 2.8-Å cocrystal structure of the Nocardia farcinica ileS T-box in complex with its cognate tRNAIle. The structure reveals a perpendicularly arranged ultrashort stem I containing a K-turn and an elongated stem II bearing an S-turn. Both stems rest against a compact pseudoknot, dock via an extended ribose zipper and jointly create a binding groove specific to the anticodon of its cognate tRNA. Contrary to proposed distal contacts to the tRNA elbow region, stem II locally reinforces the codon-anticodon interactions between stem I and tRNA, achieving low-nanomolar affinity. This study illustrates how mRNA junctions can create specific binding sites for interacting RNAs of prescribed sequence and structure.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Isoleucina-tRNA Ligase/genética , Nocardia/genética , Motivos de Nucleotídeos , RNA Bacteriano/química , RNA de Transferência de Isoleucina/química , Riboswitch/genética , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , RNA Bacteriano/metabolismo , RNA de Transferência de Isoleucina/metabolismo , Relação Estrutura-Atividade
12.
Nat Commun ; 10(1): 5542, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804502

RESUMO

Transfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp3U) is a highly conserved modification found in variable- and D-loops of tRNAs. Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.


Assuntos
Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA de Transferência/química , Uridina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Estrutura Molecular , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Uridina/genética , Uridina/metabolismo
13.
Nutrients ; 11(12)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783602

RESUMO

In animal studies, vitamin D supplementation has been shown to improve gut microbiota and intestinal inflammation. However, limited evidence exists on the effect of vitamin D supplementation on the human gut microbiota. We examined the effect of vitamin D supplementation on faecal microbiota in 26 vitamin D-deficient (25-hydroxyvitamin D (25(OH)D) ≤50 nmol/L), overweight or obese (BMI ≥25 kg/m2) otherwise healthy adults. Our study was ancillary to a community based double-blind randomised clinical trial, conducted between 2014 and 2016. The participants provided stool samples at baseline and after 100,000 international units (IU) loading dose of cholecalciferol followed by 4000 IU daily or matching placebo for 16 weeks. Faecal microbiota was analysed using 16S rRNA sequencing; V6-8 region. There was no significant difference in microbiome α-diversity between vitamin D and placebo groups at baseline and follow-up (all p > 0.05). In addition, no clustering was found based on vitamin D supplementation at follow-up (p = 0.3). However, there was a significant association between community composition and vitamin D supplementation at the genus level (p = 0.04). The vitamin D group had a higher abundance of genus Lachnospira, and lower abundance of genus Blautia (linear discriminate analysis >3.0). Moreover, individuals with 25(OH)D >75 nmol/L had a higher abundance of genus Coprococcus and lower abundance of genus Ruminococcus compared to those with 25(OH)D <50 nmol/L. Our findings suggest that vitamin D supplementation has some distinct effects on faecal microbiota. Future studies need to explore whether these effects would translate into improved clinical outcomes.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Deficiência de Vitamina D/tratamento farmacológico , Deficiência de Vitamina D/microbiologia , Vitamina D/administração & dosagem , Adulto , Bactérias/classificação , Bactérias/genética , Colecalciferol/administração & dosagem , Suplementos Nutricionais , Método Duplo-Cego , Fezes/microbiologia , Feminino , Humanos , Masculino , Obesidade/complicações , Obesidade/microbiologia , Sobrepeso/complicações , Sobrepeso/microbiologia , Placebos , RNA Bacteriano/química , RNA Ribossômico 16S/química , Análise de Sequência de RNA , Vitamina D/análogos & derivados , Vitamina D/sangue
14.
Proc Natl Acad Sci U S A ; 116(46): 23075-23082, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666318

RESUMO

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5' and 3' ends of the cloverleaf core, and these extensions get trimmed before addition of the 3'-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5' and 3' ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.


Assuntos
Escherichia coli/genética , Nucleotídeos/genética , RNA de Transferência/química , Leveduras/genética , Escherichia coli/química , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Dobramento de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Leveduras/química , Leveduras/metabolismo
15.
Nat Struct Mol Biol ; 26(12): 1106-1113, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740853

RESUMO

T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Proteínas com Domínio T/metabolismo , Aminoacilação , Proteínas de Bactérias/química , Pareamento de Bases , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA de Transferência/química , Riboswitch , Proteínas com Domínio T/química , Tuberculose/microbiologia
16.
Nat Struct Mol Biol ; 26(12): 1094-1105, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740854

RESUMO

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.


Assuntos
Aminoácidos/metabolismo , Bacillus subtilis/metabolismo , Geobacillus/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Riboswitch , Aminoacilação , Bacillus subtilis/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Geobacillus/química , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/ultraestrutura , RNA de Transferência/química , RNA de Transferência/ultraestrutura
18.
J Microbiol ; 57(10): 910-917, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31571126

RESUMO

Studies have shown that many enzymes involved in glycolysis are upregulated in Escherichia coli endoribonuclease G (rng) null mutants. However, the molecular mechanisms underlying the RNase G-associated regulation of glycolysis have not been characterized. Here, we show that RNase G cleaves the 5' untranslated region of triosephosphate isomerase A (tpiA) mRNA, leading to destabilization of the mRNA in E. coli. Nucleotide substitutions within the RNase G cleavage site in the genome resulted in altered tpiA mRNA stability, indicating that RNase G activity influences tpiA mRNA abundance. In addition, we observed that tpiA expression was enhanced, whereas that of RNase G was decreased, in E. coli cells grown anaerobically. Our findings suggest that RNase G negatively regulates tpiA mRNA abundance in response to oxygen availability in E. coli.


Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , RNA Mensageiro/genética , Triose-Fosfato Isomerase/genética , Endorribonucleases/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Triose-Fosfato Isomerase/metabolismo
19.
Microbiol Res ; 229: 126319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479952

RESUMO

Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE+ gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS- and hfq- deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE+-promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE+ mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5' mRNA translational initiation region (TIR) of the metE+ gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE+ TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE+ 5' TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE+ gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Metiltransferases/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Metiltransferases/química , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
20.
Nat Commun ; 10(1): 4304, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541094

RESUMO

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.


Assuntos
Magnésio/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Riboswitch/fisiologia , Transdução de Sinais , Xanthomonas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ligantes , Manganês/metabolismo , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/genética
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