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1.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
2.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
3.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
4.
Science ; 373(6556): 768-774, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385391

RESUMO

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Cianobactérias/química , Elementos de DNA Transponíveis , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , RNA Bacteriano/metabolismo , Transposases/química , Transposases/metabolismo
5.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298910

RESUMO

Bacterial S1 protein is a functionally important ribosomal protein. It is a part of the 30S ribosomal subunit and is also able to interact with mRNA and tmRNA. An important feature of the S1 protein family is a strong tendency towards aggregation. To study the amyloidogenic properties of S1, we isolated and purified the recombinant ribosomal S1 protein of Pseudomonas aeruginosa. Using the FoldAmyloid, Waltz, Pasta 2.0, and AGGRESCAN programs, amyloidogenic regions of the protein were predicted, which play a key role in its aggregation. The method of limited proteolysis in combination with high performance liquid chromatography and mass spectrometric analysis of the products, made it possible to identify regions of the S1 protein from P. aeruginosa that are protected from the action of proteinase K, trypsin, and chymotrypsin. Sequences of theoretically predicted and experimentally identified amyloidogenic regions were used to synthesize four peptides, three of which demonstrated the ability to form amyloid-like fibrils, as shown by electron microscopy and fluorescence spectroscopy. The identified amyloidogenic sites can further serve as a basis for the development of new antibacterial peptides against the pathogenic microorganism P. aeruginosa.


Assuntos
Amiloide/metabolismo , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Peptídeos/metabolismo , Proteólise , RNA Bacteriano/metabolismo
6.
Nat Commun ; 12(1): 4433, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290242

RESUMO

The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host.


Assuntos
Farmacorresistência Bacteriana , Helicobacter pylori/fisiologia , Lipopolissacarídeos/biossíntese , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Regiões 5' não Traduzidas , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Lipopolissacarídeos/química , Camundongos , Antígenos O/biossíntese , Antígenos O/química , Polimixina B/farmacologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Estresse Salino , Estômago/microbiologia
7.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069497

RESUMO

The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals is largely attributed to its virulence factor repertoire and the sophisticated regulatory network controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu, and to withstand Triton X-100 induced autolysis. SA564 ΔptpB mutant cells were additionally ingested faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB contributes by several ways positively to the ability of S. aureus to evade host innate immunity.


Assuntos
Neutrófilos/metabolismo , Neutrófilos/microbiologia , Infecções Estafilocócicas/imunologia , Arginina/análogos & derivados , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Peso Molecular , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Bacteriano/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismo
8.
Nat Commun ; 12(1): 3282, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078900

RESUMO

Bacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific 'universal stress responders', that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex).


Assuntos
Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , RNA Bacteriano/genética , Estresse Fisiológico/genética , Transcriptoma , Adaptação Fisiológica/genética , Atlas como Assunto , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Genes Bacterianos , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Internet , Microbiota/genética , Filogenia , RNA Bacteriano/metabolismo
9.
Nucleic Acids Res ; 49(11): 6399-6419, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096591

RESUMO

sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA-mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources.


Assuntos
Bacillus subtilis/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Aptidão Genética , Proteoma , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , Transcrição Genética
10.
J Bacteriol ; 203(14): e0001721, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33972352

RESUMO

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.


Assuntos
Firmicutes/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Esporos Bacterianos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Firmicutes/enzimologia , Firmicutes/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , Esporos Bacterianos/metabolismo
11.
Nat Commun ; 12(1): 2899, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006838

RESUMO

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.


Assuntos
Antituberculosos/administração & dosagem , Mycobacterium tuberculosis/efeitos dos fármacos , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Tuberculose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Precursores de RNA/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/genética , Resultado do Tratamento , Tuberculose/diagnóstico , Tuberculose/microbiologia
12.
Biochimie ; 187: 67-74, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34022290

RESUMO

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.


Assuntos
Escherichia coli/metabolismo , Glucose/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Estresse Fisiológico/genética
13.
Mol Cell ; 81(9): 1857-1858, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33961772

RESUMO

We talk to Ewelina Malecka and Sarah Woodson about their paper, "Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing," who inspired them along their scientific paths, the research in Sarah's lab and the environment she looks to create, as well as Ewelina's advice for aspiring scientists.


Assuntos
Pesquisa Biomédica/história , Genética/história , RNA Bacteriano/história , Pequeno RNA não Traduzido/história , Escolha da Profissão , Regulação Bacteriana da Expressão Gênica , História do Século XX , História do Século XXI , Fator Proteico 1 do Hospedeiro/metabolismo , Humanos , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Relação Estrutura-Atividade
14.
J Biol Chem ; 296: 100656, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857481

RESUMO

The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In Pseudomonas, Hfq recognizes repeats of adenine-purine-any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation-repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq-RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a 'status quo' staging point, providing nonspecific binding affinity, until Crc engages the Hfq-RNA binary complex. The dynamical components of the Hfq-RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base-phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq-RNA binding.


Assuntos
Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Motivos de Nucleotídeos , Pseudomonas aeruginosa/metabolismo , RNA Bacteriano/metabolismo , Sítios de Ligação , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/genética , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Pseudomonas aeruginosa/genética , RNA Bacteriano/química , RNA Bacteriano/genética
15.
Plant Cell ; 33(2): 248-269, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793824

RESUMO

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.


Assuntos
Membranas Intracelulares/metabolismo , Fotossíntese , Análise de Sequência de RNA , Synechocystis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multiproteicos/metabolismo , Fotossíntese/genética , Filogenia , Ligação Proteica , Biossíntese de Proteínas , Proteoma/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Tilacoides/metabolismo , Sistemas Toxina-Antitoxina , Transcriptoma/genética
16.
Plant Cell ; 33(2): 358-380, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793852

RESUMO

Phycobilisomes (PBSs), the principal cyanobacterial antenna, are among the most efficient macromolecular structures in nature, and are used for both light harvesting and directed energy transfer to the photosynthetic reaction center. However, under unfavorable conditions, excess excitation energy needs to be rapidly dissipated to avoid photodamage. The orange carotenoid protein (OCP) senses light intensity and induces thermal energy dissipation under stress conditions. Hence, its expression must be tightly controlled; however, the molecular mechanism of this regulation remains to be elucidated. Here, we describe the discovery of a posttranscriptional regulatory mechanism in Synechocystis sp. PCC 6803 in which the expression of the operon encoding the allophycocyanin subunits of the PBS is directly and in an inverse fashion linked to the expression of OCP. This regulation is mediated by ApcZ, a small regulatory RNA that is derived from the 3'-end of the tetracistronic apcABC-apcZ operon. ApcZ inhibits ocp translation under stress-free conditions. Under most stress conditions, apc operon transcription decreases and ocp translation increases. Thus, a key operon involved in the collection of light energy is functionally connected to the expression of a protein involved in energy dissipation. Our findings support the view that regulatory RNA networks in bacteria evolve through the functionalization of mRNA 3'-UTRs.


Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Luz , RNA Bacteriano/metabolismo , Synechocystis/metabolismo , Synechocystis/efeitos da radiação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Modelos Biológicos , Mutação/genética , Óperon/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Synechocystis/genética
17.
Nat Commun ; 12(1): 2085, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837194

RESUMO

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.


Assuntos
Adaptação Fisiológica/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/fisiologia , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Neoplasias Gástricas/microbiologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Carcinogênese , Modelos Animais de Doenças , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano/genética , Gerbillinae , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Interações entre Hospedeiro e Microrganismos , Humanos , Masculino , Mutação , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , RNA-Seq , Neoplasias Gástricas/patologia
18.
Nat Commun ; 12(1): 2249, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883550

RESUMO

The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.


Assuntos
Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/química , GTP Pirofosfoquinase/genética , Fator Proteico 1 do Hospedeiro/química , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Deleção de Sequência
19.
Methods Mol Biol ; 2281: 229-240, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847962

RESUMO

Single-stranded DNA-binding proteins (SSBs) are essential to all living organisms as protectors and guardians of the genome. Apart from the well-characterized RPA, humans have also evolved two further SSBs, termed hSSB1 and hSSB2. Over the last few years, we have used NMR spectroscopy to determine the molecular and structural details of both hSSBs and their interactions with DNA and RNA. Here we provide a detailed overview of how to express and purify recombinant versions of these important human proteins for the purpose of detailed structural analysis by high-resolution solution-state NMR.


Assuntos
Proteínas de Ligação a DNA/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Mitocondriais/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Clonagem Molecular , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Fermentação , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Ligação Proteica , RNA Bacteriano/metabolismo , Proteínas Recombinantes/química
20.
Methods Mol Biol ; 2263: 273-287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877603

RESUMO

Biomolecular interactions facilitate the biochemical processes that sustain life. Proteins, RNAs, and ribonucleoprotein complexes perform cellular functions that range from catalyzing the formation or cleavage of bonds to being structural building blocks, both of which are only possible through the interaction with their respective biomolecular partner(s). Having access to the parameters that describe these interactions is important for our understanding of the principles that underlie enzymatic and nonenzymatic processes. Here we describe two fluorescence-based approaches to determine two key parameters, the affinity and the rate of association/dissociation of a protein and a ligand. Considerations are provided to expand the described approach to other experimental systems.


Assuntos
Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/química , Proteínas de Ligação ao GTP/metabolismo , RNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/química , Cinética , Ligação Proteica , RNA Bacteriano/química
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