RESUMO
The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used in vivo UV crosslinking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in Salmonella enterica and Escherichia coli. Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3' end of mRNAs. Using the cspE mRNA as a model for 3' end targeting, we reveal a function for ProQ in protecting mRNA against exoribonucleolytic activity. Taken together, our results underpin the notion that ProQ governs a post-transcriptional network distinct from those of the well-characterized sRNA-binding proteins, CsrA and Hfq, and suggest a previously unrecognized, sRNA-independent role of ProQ in stabilizing mRNAs.
Assuntos
Regiões 3' não Traduzidas , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Processamento de Terminações 3' de RNA , Estabilidade de RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Salmonella enterica/metabolismo , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Exorribonucleases/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Salmonella enterica/genética , Relação Estrutura-AtividadeRESUMO
RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and noncoding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli, though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQ's FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNA's terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70, and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Aminoácidos/metabolismo , RNA Bacteriano/metabolismoRESUMO
SignificanceWhile most small, regulatory RNAs are thought to be "noncoding," a few have been found to also encode a small protein. Here we describe a 164-nucleotide RNA that encodes a 28-amino acid, amphipathic protein, which interacts with aerobic glycerol-3-phosphate dehydrogenase and increases dehydrogenase activity but also base pairs with two mRNAs to reduce expression. The coding and base-pairing sequences overlap, and the two regulatory functions compete.
Assuntos
Carbono/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/fisiologia , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Galactose/metabolismo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismoRESUMO
The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Mutagênese , Protease La/metabolismo , Controle de Qualidade , RNA Bacteriano/genética , Proteínas de Ligação a RNA/metabolismo , Salmonella enterica/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a RNA/genética , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimentoRESUMO
Avian pathogenic Escherichia coli (APEC) is a notable subpathotype of the nonhuman extraintestinal pathogenic E. coli (ExPEC). Recognized as an extraintestinal foodborne pathogen, the zoonotic potential of APEC/ExPEC allows for cross-host transmission via APEC-contaminated poultry meat and eggs. ProQ, an RNA binding protein, is evolutionarily conserved in E. coli. However, its regulatory roles in the biofilm formation and virulence of APEC/ExPEC have not been explored. In this study, proQ deletion in the APEC strain FY26 significantly compromised its biofilm-forming ability. Furthermore, animal tests and cellular infection experiments showed that ProQ depletion significantly attenuated APEC virulence, thereby diminishing its capacity for bloodstream infection and effective adherence to and persistence within host cells. Transcriptome analysis revealed a decrease in the transcription level of the small RNA (sRNA) RyfA in the mutant FY26ΔproQ, suggesting a direct interaction between the sRNA RyfA and ProQ. This interaction might indicate that sRNA RyfA is a novel ProQ-associated sRNA. Moreover, the direct binding of ProQ to the sRNA RyfA was crucial for APEC biofilm formation, pathogenicity, adhesion, and intracellular survival. In conclusion, our findings provide detailed insight into the interaction between ProQ and sRNA RyfA and deepen our understanding of the regulatory elements that dictate APEC virulence and biofilm development. Such insights are instrumental in developing strategies to counteract APEC colonization within hosts and impede APEC biofilm establishment on food surfaces.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Doenças das Aves Domésticas , Pequeno RNA não Traduzido , Animais , Escherichia coli , Virulência , Infecções por Escherichia coli/veterinária , Galinhas/genética , Doenças das Aves Domésticas/patologia , Fatores de Virulência/genética , Biofilmes , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação a RNARESUMO
The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.
Assuntos
Proteínas de Escherichia coli , Pasteurella multocida , Pequeno RNA não Traduzido , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Small regulatory RNAs (sRNAs) play an important role for posttranscriptional gene regulation in bacteria. sRNAs recognize their target messenger RNAs (mRNAs) by base-pairing, which is often facilitated by interactions with the bacterial RNA-binding proteins Hfq or ProQ. The FinO/ProQ RNA-binding protein domain was first discovered in the bacterial repressor of conjugation, FinO. Since then, the functional role of FinO/ProQ-like proteins in posttranscriptional gene regulation was extensively studied in particular in the enterobacteria E. coli and Salmonella enterica and a wide range of sRNA-targets was identified for these proteins. In addition, enterobacterial ProQ homologs also recognize and protect the 3'-ends of a number of mRNAs from exonucleolytic degradation. However, the RNA-binding properties of FinO/ProQ proteins with regard to the recognition of different RNA targets are not yet fully understood. Here, we present the solution NMR structure of the so far functionally uncharacterized ProQ homolog Lpp1663 from Legionella pneumophila as a newly confirmed member and a minimal model system of the FinO/ProQ protein family. In addition, we characterize the RNA-binding preferences of Lpp1663 with high resolution NMR spectroscopy and isothermal titration calorimetry (ITC). Our results suggest a binding preference for single-stranded uridine-rich RNAs in the vicinity of stable stem-loop structures. According to chemical shift perturbation experiments, the single-stranded U-rich RNAs interact mainly with a conserved RNA-binding surface on the concave site of Lpp1663.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Legionella pneumophila/genética , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , Relação Estrutura-AtividadeRESUMO
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/metabolismoRESUMO
Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in many economically important plants, including apples and pears. This bacterium produces three exopolysaccharides (EPSs), amylovoran, levan, and cellulose, and forms biofilms in host plant vascular tissues, which are crucial for pathogenesis. Here, we demonstrate that ProQ, a conserved bacterial RNA chaperone, was required for the virulence of E. amylovora in apple shoots and for biofilm formation in planta. In vitro experiments revealed that the deletion of proQ increased the production of amylovoran and cellulose. Prc is a putative periplasmic protease, and the prc gene is located adjacent to proQ. We found that Prc and the associated lipoprotein NlpI negatively affected amylovoran production, whereas Spr, a peptidoglycan hydrolase degraded by Prc, positively regulated amylovoran. Since the prc promoter is likely located within proQ, our data showed that proQ deletion significantly reduced the prc mRNA levels. We used a genome-wide transposon mutagenesis experiment to uncover the involvement of the bacterial second messenger c-di-GMP in ProQ-mediated cellulose production. The deletion of proQ resulted in elevated intracellular c-di-GMP levels and cellulose production, which were restored to wild-type levels by deleting genes encoding c-di-GMP biosynthesis enzymes. Moreover, ProQ positively affected the mRNA levels of genes encoding c-di-GMP-degrading phosphodiesterase enzymes via a mechanism independent of mRNA decay. In summary, our study revealed a detailed function of E. amylovora ProQ in coordinating cellulose biosynthesis and, for the first time, linked ProQ with c-di-GMP metabolism and also uncovered a role of Prc in the regulation of amylovoran production. IMPORTANCE Fire blight, caused by the bacterium Erwinia amylovora, is an important disease affecting many rosaceous plants, including apple and pear, that can lead to devastating economic losses worldwide. Similar to many xylem-invading pathogens, E. amylovora forms biofilms that rely on the production of exopolysaccharides (EPSs). In this paper, we identified the RNA-binding protein ProQ as an important virulence regulator. ProQ played a central role in controlling the production of EPSs and participated in the regulation of several conserved bacterial signal transduction pathways, including the second messenger c-di-GMP and the periplasmic protease Prc-mediated systems. Since ProQ has recently been recognized as a global posttranscriptional regulator in many bacteria, these findings provide new insights into multitiered regulatory mechanisms for the precise control of virulence factor production in bacterial pathogens.
Assuntos
Erwinia amylovora , Malus , Pyrus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulose/metabolismo , Erwinia amylovora/metabolismo , Malus/microbiologia , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Pyrus/microbiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sistemas do Segundo MensageiroRESUMO
In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences. In bacteria, three main proteins - Hfq, ProQ, and CsrA - have been shown to regulate numerous complex processes, including bacterial growth, stress response and virulence. Hfq and ProQ have well-studied activities as global chaperones with pleiotropic impact, while CsrA has a chaperone-like role with more defined riboregulatory function. Here, we describe relevant novel insights into their common features, including RNA binding properties, unstructured domains, and interplay with other proteins important to RNA metabolism.
Assuntos
RNA Bacteriano , Pequeno RNA não Traduzido , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode-of-action of RaiZ, a ProQ-dependent sRNA that is made from the 3' end of the mRNA encoding ribosome-inactivating protein RaiA. We show that RaiZ is a base-pairing sRNA that represses in trans the mRNA of histone-like protein HU-α. RaiZ forms an RNA duplex with the ribosome-binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU-α. Similarities and differences between ProQ- and Hfq-mediated regulation will be discussed.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas de Transporte/biossíntese , Proteínas de Membrana Transportadoras/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Salmonella typhimurium/metabolismo , Proteínas de Membrana Transportadoras/genética , RNA Bacteriano/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Pequeno RNA não Traduzido/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Salmonella typhimurium/genéticaRESUMO
It is now established that base-pairing regulatory RNAs are key players in post-transcriptional regulatory networks where they affect the translation and/or stability of their target RNAs. In many cases, the base-pairing between two RNAs is facilitated by an RNA-binding protein (RBP) that serves as an RNA chaperone. Recent advances in sequencing methods have revealed the RNA populations bound by the RBPs, yielding insights valuable into regulatory networks. Further analyses of these networks can improve our understanding of the roles played by RBPs in the regulation of gene expression by regulatory RNAs, especially when multiple RBPs are involved. For example, using an RNA sequencing-based methodology that captures RNA-RNA pairs on RBP, an interplay between two RBPs in bacteria that compete on the same RNA-RNA pair was revealed. In this case, one protein promotes negative regulation of the target RNA, while the second protein can block this regulation. In this mini-review, I outline the exciting future directions that can be taken to deepen our understanding of the roles played by RBPs in post-transcriptional regulation, and discuss how the different sequencing methods can assist in deciphering the relationships among RBPs, and between the RBPs and the RNAs they bind. Having a more detailed picture of the RBPs-RNAs network will elucidate how bacteria can have nuanced control of gene expression, critical for survival in the varied environments in which bacteria live.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genéticaRESUMO
BACKGROUND: To respond and adapt to environmental challenges, prokaryotes regulate cellular processes rapidly and reversibly through protein phosphorylation and dephosphorylation. This study investigates the intracellular proteome and Ser/Thr/Tyr phosphoproteome of the oral commensal Streptococcus gordonii. Intracellular proteins from planktonic cells of S. gordonii DL1 were extracted and subjected to 2D-gel electrophoresis. Proteins in general were visualized using Coomassie Brilliant Blue and T-Rex staining. Phosphorylated proteins were visualized with Pro-Q Diamond Phosphoprotein Gel Stain. Proteins were identified by LC-MS/MS and sequence analysis. RESULTS: In total, sixty-one intracellular proteins were identified in S. gordonii DL1, many of which occurred at multiple isoelectric points. Nineteen of these proteins were present as one or more Ser/Thr/Tyr phosphorylated form. The identified phosphoproteins turned out to be involved in a variety of cellular processes. CONCLUSION: Nineteen phosphoproteins involved in various cellular functions were identified in S. gordonii. This is the first time the global intracellular Ser/Thr/Tyr phosphorylation profile has been analysed in an oral streptococcus. Comparison with phosphoproteomes of other species from previous studies showed many similarities. Proteins that are consistently found in a phosphorylated state across several species and growth conditions may represent a core phosphoproteome profile shared by many bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Fosfoproteínas/metabolismo , Streptococcus gordonii/metabolismo , Proteínas de Bactérias/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Humanos , Boca/microbiologia , Fosfoproteínas/análise , Fosforilação , Serina/metabolismo , Streptococcus gordonii/isolamento & purificação , Espectrometria de Massas em Tandem , Treonina/metabolismo , Tirosina/metabolismoRESUMO
Post-transcriptional gene regulation in bacteria plays a major role in the adaptation of bacterial cells to the changing conditions encountered in the environment. In bacteria, most of the regulation at the level of mRNA seems to be targeting the 5'untranslated regions where accessibility to the ribosome-binding site can be modulated to alter gene expression. In recent years, the role of 3'untranslated regions has gained attention also as a site for post-transcriptional regulation. In addition to be a source of trans-encoded small RNAs, the 3'untranslated regions can be targets to modulate gene expression. Taking recent findings in the post-transcriptional regulation of the hilD gene, encoding for the main regulator of virulence in Salmonella enterica serovar Typhimurium, we highlight the role of 3'untranslated regions as targets of post-transcriptional regulation mediated by small RNAs and discuss the implications of transcriptional elongation in the 3'UTR-mediated regulation in bacteria.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , Proteínas de Bactérias/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência/genéticaRESUMO
The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia coli ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismoRESUMO
A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.
Assuntos
Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , Legionella pneumophila/genética , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Perfilação da Expressão Gênica/métodos , Humanos , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Modelos Genéticos , Regulon/genética , Transformação BacterianaRESUMO
The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA-protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.
Assuntos
Proteínas de Bactérias/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Salmonella enterica/metabolismo , Sequência Conservada/genética , Ligantes , Análise de Componente Principal , RNA Bacteriano/genética , RNA não Traduzido/genéticaRESUMO
Protein phosphorylation, one of the most important post-translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al3+ ) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non-phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA-Al3+ -phosphoprotein complex, unlike non-phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro-Q Diamond stain. ACRA stain can detect 1-2 ng of α-casein and ß-casein, 8-16 ng of ovalbumin (OVA) and κ-casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro-Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8-500 ng, correlation coefficient r = 0.999), α-casein (4-500 ng, r = 0.992), ß-casein (4-500 ng, r = 0.996), and κ-casein (8-500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.
Assuntos
Antracenos/química , Corantes/química , Eletroforese em Gel de Poliacrilamida/métodos , Fosfoproteínas/análise , Coloração e Rotulagem/métodos , Antracenos/análise , Corantes/análise , Modelos Lineares , Fosfoproteínas/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Glicoproteínas/análise , Hidrazinas/química , Avidina/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional/métodos , Corantes Fluorescentes/síntese química , Humanos , Hidrazinas/síntese química , Orosomucoide/análise , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Transferrina/análiseRESUMO
A sensitive and simple technique was developed for the visualization of gel-separated lipopolysaccharides by using a hydrazide derivative, UGF202. As low as 0.5-1 ng total LPS could be detected by UGF202 stain, which is 2- and 16-fold more sensitive than that of the commonly used Pro-Q Emerald 300 and Keenan et al. developed silver stain, respectively. The results indicated that UGF202 stain could be a good choice for LPS determination in polyacrylamide gels.