RESUMO
Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes). These mechanisms can allow specific destruction of a regulatory RNA via pairing with a decay trigger RNA or function as global off switches by disrupting the stability or function of the protein partner.
Assuntos
MicroRNAs , Estabilidade de RNA , MicroRNAs/metabolismo , MicroRNAs/genética , Estabilidade de RNA/genética , Animais , Humanos , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Regulação da Expressão GênicaRESUMO
Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfqâ DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfqâ DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfqâ DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.
Assuntos
Proteínas de Escherichia coli , Pequeno RNA não Traduzido , Proteínas de Bactérias/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , Pareamento de Bases , RNA Bacteriano/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , Domínio Catalítico/genética , Endorribonucleases/genética , Exorribonucleases/genética , Regulação Bacteriana da Expressão Gênica/genética , Chaperonas Moleculares/genética , Estabilidade de RNA/genética , Pequeno RNA não Traduzido/genéticaRESUMO
Bacterial small RNAs (sRNAs) regulate the expression of hundreds of transcripts via base pairing mediated by the Hfq chaperone protein. sRNAs and the mRNA sites they target are heterogeneous in sequence, length, and secondary structure. To understand how Hfq can flexibly match diverse sRNA and mRNA pairs, we developed a single-molecule Förster resonance energy transfer (smFRET) platform that visualizes the target search on timescales relevant in cells. Here we show that unfolding of target secondary structure on Hfq creates a kinetic energy barrier that determines whether target recognition succeeds or aborts before a stable anti-sense complex is achieved. Premature dissociation of the sRNA can be alleviated by strong RNA-Hfq interactions, explaining why sRNAs have different target recognition profiles. We propose that the diverse sequences and structures of Hfq substrates create an additional layer of information that tunes the efficiency and selectivity of non-coding RNA regulation in bacteria.
Assuntos
Escherichia coli K12/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Cinética , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Estabilidade de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Análise de Célula Única , Relação Estrutura-AtividadeRESUMO
Small regulatory RNA (sRNAs) are key mediators of posttranscriptional gene control in bacteria. Assisted by RNA-binding proteins, a single sRNA often modulates the expression of dozens of genes, and thus sRNAs frequently adopt central roles in regulatory networks. Posttranscriptional regulation by sRNAs comes with several unique features that cannot be achieved by transcriptional regulators. However, for optimal network performance, transcriptional and posttranscriptional control mechanisms typically go hand-in-hand. This view is reflected by the ever-growing class of mixed network motifs involving sRNAs and transcription factors, which are ubiquitous in biology and whose regulatory properties we are beginning to understand. In addition, sRNA activity can be antagonized by base-pairing with sponge RNAs, adding yet another layer of complexity to these networks. In this article, we summarize the regulatory concepts underlying sRNA-mediated gene control in bacteria and discuss how sRNAs shape the output of a network, focusing on several key examples.
Assuntos
RNA Bacteriano , Pequeno RNA não Traduzido , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Regulon , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias/genética , Bactérias/metabolismo , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismoRESUMO
The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.
Assuntos
Clostridioides difficile , Clostridioides , Animais , Humanos , Camundongos , Clostridioides/genética , Clostridioides/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Antibacterianos , RNA/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
The widely occurring bacterial RNA chaperone Hfq is a key factor in the post-transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo-EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA-specific quaternary architectures resulting from the combination of multivalent protein-protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold.
Assuntos
Proteínas de Bactérias , Ribonucleoproteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismoRESUMO
The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.
Assuntos
Klebsiella pneumoniae , Pequeno RNA não Traduzido , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Pequeno RNA não Traduzido/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Divisão Celular/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.
Assuntos
Pseudomonas aeruginosa , Pequeno RNA não Traduzido , Pseudomonas aeruginosa/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , RNA Mensageiro/genética , Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genéticaRESUMO
Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.
Assuntos
Proteínas de Escherichia coli , Pequeno RNA não Traduzido , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismoRESUMO
Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans-encoded transcripts and are typically non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual-function RNAs. In this study, we discovered a dual-function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae.
Assuntos
Carbono/metabolismo , Toxina da Cólera/metabolismo , Citrato (si)-Sintase/metabolismo , RNA Bacteriano/genética , Vibrio cholerae/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Testes Genéticos , Fases de Leitura Aberta , Fenótipo , RNA Bacteriano/metabolismo , Vibrio cholerae/genéticaRESUMO
Initiation is the rate-limiting step of translation, and in bacteria, mRNA secondary structure has been extensively reported as limiting the efficiency of translation by occluding the ribosome-binding site. In striking contrast with this inhibitory effect, we report here that stem-loop structures located within coding sequences instead activate translation initiation of the Escherichia coli fepA and bamA mRNAs involved in iron acquisition and outer membrane proteins assembly, respectively. Both structures promote ribosome binding in vitro, independently of their nucleotide sequence. Moreover, two small regulatory RNAs, OmrA and OmrB, base pair to and most likely disrupt the fepA stem-loop structure, thereby repressing FepA synthesis. By expanding our understanding of how mRNA cis-acting elements regulate translation, these data challenge the widespread view of mRNA secondary structures as translation inhibitors and show that translation-activating elements embedded in coding sequences can be targeted by small RNAs to inhibit gene expression.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Receptores de Superfície Celular/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Pareamento de Bases , Sequência de Bases , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Sequências Repetidas Invertidas , Ferro/metabolismo , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Receptores de Superfície Celular/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.
Assuntos
Proteínas de Bactérias/metabolismo , Endorribonucleases/metabolismo , Precursores de RNA/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Salmonella enterica/enzimologia , Regiões 3' não Traduzidas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Biologia Computacional , Bases de Dados Genéticas , Endorribonucleases/química , Endorribonucleases/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Salmonella enterica/genética , Relação Estrutura-Atividade , Transcriptoma , Uridina/metabolismoRESUMO
The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq-facilitated sRNA-mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3'-5'-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA-mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.
Assuntos
Endorribonucleases , Escherichia coli , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Motivos de Ligação ao RNA , RNA Mensageiro/metabolismo , RNA/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 small RNA (sRNA) RydC strongly activates cfa, which encodes the cyclopropane fatty acid synthase. Previous work demonstrated that RydC activation of cfa increases the conversion of unsaturated fatty acids to cyclopropanated fatty acids in membrane lipids and changes the biophysical properties of membranes, making cells more resistant to acid stress. The regulators that control RydC synthesis had not previously been identified. In this study, we identify a GntR-family transcription factor, YieP, that represses rydC transcription. YieP positively autoregulates its own transcription and indirectly regulates cfa through RydC. We further identify additional sRNA regulatory inputs that contribute to the control of RydC and cfa. The translation of yieP is repressed by the Fnr-dependent sRNA, FnrS, making FnrS an indirect activator of rydC and cfa. Conversely, RydC activity on cfa is antagonized by the OmpR-dependent sRNA OmrB. Altogether, this work illuminates a complex regulatory network involving transcriptional and post-transcriptional inputs that link the control of membrane biophysical properties to multiple environmental signals. IMPORTANCE: Bacteria experience many environmental stresses that challenge their membrane integrity. To withstand these challenges, bacteria sense what stress is occurring and mount a response that protects membranes. Previous work documented the important roles of small RNA (sRNA) regulators in membrane stress responses. One sRNA, RydC, helps cells cope with membrane-disrupting stresses by promoting changes in the types of lipids incorporated into membranes. In this study, we identified a regulator, YieP, that controls when RydC is produced and additional sRNA regulators that modulate YieP levels and RydC activity. These findings illuminate a complex regulatory network that helps bacteria sense and respond to membrane stress.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Transcrição Gênica , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintases/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ciclopropanos , Ácidos Graxos , MetiltransferasesRESUMO
Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery-facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design-even though its main intracellular transportation mode is free diffusion.
Assuntos
Escherichia coli , Ribossomos , Escherichia coli/genética , Ribossomos/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA/metabolismo , RNA Mensageiro/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.
Assuntos
Haemophilus somnus , Pasteurellaceae , Animais , Bovinos , Camundongos , Virulência/genética , Haemophilus somnus/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas/metabolismo , Monócitos , Pasteurellaceae/genéticaRESUMO
Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an â¼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol.
Assuntos
Regiões 3' não Traduzidas , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Estresse Psicológico , Bactérias/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sequência Conservada , Endorribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Potenciais da Membrana , Proteínas de Membrana/genética , Viabilidade Microbiana , Dados de Sequência Molecular , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Transdução de Sinais , Fatores de TempoRESUMO
As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3' to 5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.