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1.
Nucleic Acids Res ; 51(18): 9804-9820, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37650646

RESUMO

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2-5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5' terminus of RNA substrates, NrnB acts at the 3' terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3'-5' exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Exorribonucleases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Fosfodiesterase I , RNA/metabolismo
2.
Nucleic Acids Res ; 50(21): 12369-12388, 2022 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-36478094

RESUMO

Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.


Assuntos
Exonucleases , Firmicutes , RNA , Proteínas de Bactérias/metabolismo , Exonucleases/química , Nucleotídeos , RNA/metabolismo , Firmicutes/química , Firmicutes/classificação , Firmicutes/enzimologia
3.
Mol Cell ; 57(6): 1088-1098, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25794617

RESUMO

Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory ("metalloregulatory") proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni(2+) and Co(2+). These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co(2+)-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobalto/metabolismo , Níquel/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Riboswitch/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte de Cátions/genética , Clostridium/genética , Clostridium/metabolismo , Sequência Conservada , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico
4.
Mol Microbiol ; 114(2): 348-360, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32314426

RESUMO

The ANTAR domain harnesses RNA-binding activity to promote transcription attenuation. Although several ANTAR proteins have been analyzed by high-resolution structural analyses, the residues involved in RNA-recognition and transcription attenuation have not been identified. Nor is it clear how signal-responsive domains are allosterically coupled with ANTAR domains for control of gene expression. Herein, we examined the sequence conservation of ANTAR domains to find residues that may associate with RNA. We subjected the corresponding positions of Klebsiella oxytoca NasR to site-directed alanine substitutions and measured RNA-binding activity. This revealed a functionally important patch of residues that forms amino acid pairing interactions with residues from NasR's nitrate-sensing NIT domain. We hypothesize these amino acid pairing interactions are part of an autoinhibitory mechanism that holds the structure in an "off" state in the absence of nitrate signal. Indeed, mutational disruption of these interactions resulted in constitutively active proteins, freed from autoinhibition and no longer influenced by nitrate. Moreover, sequence analyses suggested the autoinhibitory mechanism has been evolutionarily maintained by NasR proteins. These data reveal a molecular mechanism for how NasR couples its nitrate signal to RNA-binding activity, and generally show how signal-responsive domains of one-component regulatory proteins have evolved to exert control over RNA-binding ANTAR domains.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Klebsiella oxytoca/genética , Klebsiella oxytoca/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Óperon/genética , RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Motivos de Ligação ao RNA/genética , Transativadores/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética
5.
J Bacteriol ; 201(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31138629

RESUMO

The synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescent yfp reporter based on a c-di-GMP-responsive riboswitch to visualize the relative abundance of c-di-GMP for single cells of the Gram-positive model organism Bacillus subtilis Coupled with cell-type-specific fluorescent reporters, this riboswitch reporter revealed that c-di-GMP levels are markedly different among B. subtilis cellular subpopulations. For example, cells that have made the decision to become matrix producers maintain higher intracellular c-di-GMP concentrations than motile cells. Similarly, we find that c-di-GMP levels differ between sporulating and competent cell types. These results suggest that biochemical measurements of c-di-GMP abundance are likely to be inaccurate for a bulk ensemble of B. subtilis cells, as such measurements will average c-di-GMP levels across the population. Moreover, the significant variation in c-di-GMP levels between cell types hints that c-di-GMP might play an important role during B. subtilis biofilm formation. This study therefore emphasizes the importance of using single-cell approaches for analyzing metabolic trends within ensemble bacterial populations.IMPORTANCE Many bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities. Bacillus subtilis is a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for single B. subtilis cells. Our analysis shows that c-di-GMP levels are conspicuously different among B. subtilis cellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.


Assuntos
Bacillus subtilis/citologia , GMP Cíclico/análogos & derivados , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/análise , GMP Cíclico/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia , Análise de Célula Única
6.
J Bacteriol ; 201(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30833356

RESUMO

Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR).IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.


Assuntos
Repressão Catabólica , Enterococcus faecalis/metabolismo , Etanolamina/metabolismo , Regulação Bacteriana da Expressão Gênica , Meios de Cultura/química , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Deleção de Genes , Genes Reguladores , Teste de Complementação Genética
7.
J Bacteriol ; 200(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30249708

RESUMO

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in Pseudomonas aeruginosa as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of orn accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an orn ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from Vibrio cholerae and Bacillus anthracis, we found that only enzymes known to cleave oligoribonucleotides (orn and nrnA) rescued the P. aeruginosa Δorn mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (nrnA, nrnB, nrnC, and orn) can also hydrolyze pGpG. A B. subtilisnrnA nrnB mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides.IMPORTANCE The bacterial bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3'-to-5' exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in Pseudomonas aeruginosa Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of Bacillus anthracis and Vibrio cholerae that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.


Assuntos
Bacillus anthracis/enzimologia , GMP Cíclico/análogos & derivados , Exorribonucleases/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Exorribonucleases/genética , Hidrólise , Mutação , Pseudomonas aeruginosa/enzimologia , Sistemas do Segundo Mensageiro , Transdução de Sinais
9.
PLoS Genet ; 10(6): e1004429, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24968120

RESUMO

Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5-2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.


Assuntos
Transporte Biológico/genética , Proteínas de Transporte de Cátions/genética , Magnésio/metabolismo , Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Transporte de Cátions/isolamento & purificação , Proteínas de Transporte de Cátions/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Riboswitch/genética
10.
Biochim Biophys Acta ; 1839(10): 989-994, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24769284

RESUMO

Catalysis in the biological context was largely thought to be a protein-based phenomenon until the discovery of RNA catalysts called ribozymes. These discoveries demonstrated that many RNA molecules exhibit remarkable structural and functional versatility. By virtue of these features, naturally occurring ribozymes have been found to be involved in catalyzing reactions for fundamentally important cellular processes such as translation and RNA processing. Another class of RNAs called riboswitches directly binds ligands to control downstream gene expression. Most riboswitches regulate downstream gene expression by controlling premature transcription termination or by affecting the efficiency of translation initiation. However, one riboswitch class couples ligand-sensing to ribozyme activity. Specifically, the glmS riboswitch is a nucleolytic ribozyme, whose self-cleavage activity is triggered by the binding of GlcN6P. The products of this self-cleavage reaction are then targeted by cellular RNases for rapid degradation, thereby reducing glmS expression under conditions of sufficient GlcN6P. Since the discovery of the glmS ribozyme, other metabolite-binding ribozymes have been identified. Together, these discoveries have expanded the general understanding of noncoding RNAs and provided insights that will assist future development of synthetic riboswitch-ribozymes. A very broad overview of natural and synthetic ribozymes is presented herein with an emphasis on the structure and function of the glmS ribozyme as a paradigm for metabolite-binding ribozymes that control gene expression. This article is part of a Special Issue entitled: Riboswitches.

11.
PLoS Genet ; 8(6): e1002666, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22685413

RESUMO

ANTAR proteins are widespread bacterial regulatory proteins that have RNA-binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.


Assuntos
Enterococcus faecalis , Nitrogênio/metabolismo , Proteínas de Ligação a RNA/genética , RNA , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Fosforilação , Filogenia , Multimerização Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/genética , Dobramento de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais
12.
J Bacteriol ; 196(6): 1206-14, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24415722

RESUMO

Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions.


Assuntos
Antiporters/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Magnésio/metabolismo , Antiporters/genética , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Técnicas de Inativação de Genes , Supressão Genética
13.
Structure ; 32(9): 1488-1497.e5, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38959899

RESUMO

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding ß-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family.


Assuntos
Proteínas de Bactérias , Ligação Proteica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Sítios de Ligação , Modelos Moleculares , Thermoanaerobacter/metabolismo , Thermoanaerobacter/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , Conformação de Ácido Nucleico , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética
14.
Cell Rep ; 43(9): 114759, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39276351

RESUMO

RNA degradation is a central process required for transcriptional regulation. Eventually, this process degrades diribonucleotides into mononucleotides by specific diribonucleases. In Escherichia coli, oligoribonuclease (Orn) serves this function and is unique as the only essential exoribonuclease. Yet, related organisms, such as Pseudomonas aeruginosa, display a growth defect but are viable without Orn, contesting its essentiality. Here, we take advantage of P. aeruginosa orn mutants to screen for suppressors that restore colony morphology and identified yciV. Purified YciV (RNase AM) exhibits diribonuclease activity. While RNase AM is present in all γ-proteobacteria, phylogenetic analysis reveals differences that map to the active site. RNase AMPa expression in E. coli eliminates the necessity of orn. Together, these results show that diribonuclease activity prevents toxic diribonucleotide accumulation in γ-proteobacteria, suggesting that diribonucleotides may be utilized to monitor RNA degradation efficacy. Because higher eukaryotes encode Orn, these observations indicate a conserved mechanism for monitoring RNA degradation.


Assuntos
Exorribonucleases , Exorribonucleases/metabolismo , Exorribonucleases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Estabilidade de RNA , Filogenia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mutação/genética
15.
Microbiol Spectr ; : e0335722, 2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36786617

RESUMO

The shell proteins that comprise bacterial microcompartments (BMCs) can self-assemble into an array of superstructures such as nanotubes, flat sheets, and icosahedra. The physical characterization of BMCs and these superstructures typically relies on electron microscopy, which decouples samples from their solution context. We hypothesize that an investigation of fluorescently tagged BMCs and shell protein superstructures in vitro using high-resolution confocal microscopy will lead to new insights into the solution behavior of these entities. We find that confocal imaging is able to capture nanotubes and sheets previously reported by transmission electron microscopy (TEM). Using a combination of fluorescent tags, we present qualitative evidence that these structures intermix with one another in a hetero- and homotypic fashion. Complete BMCs are also able to accomplish intermixing as evidenced by colocalization data. Finally, a simple colocalization experiment suggests that fluorescently modified encapsulation peptides (EPs) may prefer certain shell protein binding partners. Together, these data demonstrate that high-resolution confocal microscopy is a powerful tool for investigating microcompartment-related structures in vitro, particularly for colocalization analyses. These results also support the notion that BMCs may intermix protein components, presumably from the outer shell. IMPORTANCE Microcompartments are large, organelle-like structures that help bacteria catabolize targeted metabolites while also protecting the cytosol against highly reactive metabolic intermediates. Their protein shell self-assembles into a polyhedral structure of approximately 100 to 200 nm in diameter. Inside the shell are thousands of copies of cargo enzymes, which are responsible for a specific metabolic pathway. While different approaches have revealed high-resolution structures of individual microcompartment proteins, it is less clear how these factors self-assemble to form the full native structure. In this study, we show that laser scanning confocal microscopy can be used to study microcompartment proteins. We find that this approach allows researchers to investigate the interactions and potential exchange of shell protein subunits in solution. From this, we conclude that confocal microscopy offers advantages for studying the in vitro structures of other microcompartments as well as carboxysomes and other bacterial organelles.

16.
Nat Struct Mol Biol ; 14(4): 308-17, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17384645

RESUMO

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5' untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ(1)), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ(1) aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ(1) in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.


Assuntos
Aptâmeros de Nucleotídeos/química , Bacillus subtilis/genética , Nucleosídeo Q/metabolismo , Pirimidinonas/metabolismo , Pirróis/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Regiões 5' não Traduzidas/genética , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases/genética , Sequência de Bases , Sequência Conservada , Diálise , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Nucleosídeo Q/química , Filogenia , Pirimidinonas/química , Pirróis/química , RNA Bacteriano/química , RNA Bacteriano/genética
17.
Nucleic Acids Res ; 38(19): 6637-51, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20525796

RESUMO

Post-transcriptional regulatory mechanisms are widespread in bacteria. Interestingly, current published data hint that some of these mechanisms may be non-random with respect to their phylogenetic distribution. Although small, trans-acting regulatory RNAs commonly occur in bacterial genomes, they have been better characterized in Gram-negative bacteria, leaving the impression that they may be less important for Firmicutes. It has been presumed that Gram-positive bacteria, in particular the Firmicutes, are likely to utilize cis-acting regulatory RNAs located within the 5' mRNA leader region more often than trans-acting regulatory RNAs. In this analysis we catalog, by a deep sequencing-based approach, both classes of regulatory RNA candidates for Bacillus subtilis, the model microorganism for Firmicutes. We successfully recover most of the known small RNA regulators while also identifying a greater number of new candidate RNAs. We anticipate these data to be a broadly useful resource for analysis of post-transcriptional regulatory strategies in B. subtilis and other Firmicutes.


Assuntos
Bacillus subtilis/genética , Pequeno RNA não Traduzido/análise , Regiões 5' não Traduzidas , Toxinas Bacterianas/genética , Sequência de Bases , Dados de Sequência Molecular , Prófagos/genética , RNA Antissenso/análise , RNA Bacteriano/análise , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Análise de Sequência de RNA
18.
Proc Natl Acad Sci U S A ; 106(11): 4435-40, 2009 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-19246383

RESUMO

Ethanolamine, a product of the breakdown of phosphatidylethanolamine from cell membranes, is abundant in the human intestinal tract and in processed foods. Effective utilization of ethanolamine as a carbon and nitrogen source may provide a survival advantage to bacteria that inhabit the gastrointestinal tract and may influence the virulence of pathogens. In this work, we describe a unique series of posttranscriptional regulatory strategies that influence expression of ethanolamine utilization genes (eut) in Enterococcus, Clostridium, and Listeria species. One of these mechanisms requires an unusual 2-component regulatory system. Regulation involves specific sensing of ethanolamine by a sensor histidine kinase (EutW), resulting in autophosphorylation and subsequent phosphoryl transfer to a response regulator (EutV) containing a RNA-binding domain. Our data suggests that EutV is likely to affect downstream gene expression by interacting with conserved transcription termination signals located within the eut locus. Breakdown of ethanolamine requires adenosylcobalamin (AdoCbl) as a cofactor, and, intriguingly, we also identify an intercistronic AdoCbl riboswitch that has a predicted structure different from previously established AdoCbl riboswitches. We demonstrate that association of AdoCbl to this riboswitch prevents formation of an intrinsic transcription terminator element located within the intercistronic region. Together, these results suggest an intricate and carefully coordinated interplay of multiple regulatory strategies for control of ethanolamine utilization genes. Gene expression appears to be directed by overlapping posttranscriptional regulatory mechanisms, each responding to a particular metabolic signal, conceptually akin to regulation by multiple DNA-binding transcription factors.


Assuntos
Etanolamina/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/genética , Proteínas de Bactérias , Clostridium , Enterococcus , Enterococcus faecalis/metabolismo , Trato Gastrointestinal/microbiologia , Histidina Quinase , Humanos , Listeria , Proteínas Quinases/fisiologia , Fatores de Transcrição
19.
Trends Biochem Sci ; 32(9): 415-24, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17764952

RESUMO

Riboswitches, metabolite-sensing RNA elements that are present in untranslated regions of the transcripts that they regulate, possess extensive tertiary structure to couple metabolite binding to genetic control. Here we discuss recently published structures from four riboswitch classes and compare these natural RNA structures to those of in-vitro-selected RNA aptamers, which bind ligands similar to those of the riboswitches. In addition, we examine the glmS riboswitch - the first example of a ribozyme-based riboswitch. This RNA provides the latest twist in the riboswitch field and portends exciting advances in the coming years. Our knowledge of the mechanisms underlying genetic regulation by riboswitches has increased mightily in recent years and will continue to grow as new riboswitch classes and ligands are discovered and structurally characterized.


Assuntos
Regulação da Expressão Gênica , RNA Catalítico , Ribossomos/fisiologia , Animais , Pareamento de Bases , Sequência de Bases , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico
20.
FEMS Microbiol Rev ; 46(6)2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36026528

RESUMO

Diribonucleotides arise from two sources: turnover of RNA transcripts (rRNA, tRNA, mRNA, and others) and linearization of cyclic-di-nucleotide signaling molecules. In both cases, there appears to be a requirement for a dedicated set of enzymes that will cleave these diribonucleotides into mononucleotides. The first enzyme discovered to mediate this activity is oligoribonuclease (Orn) from Escherichia coli. In addition to being the enzyme that cleaves dinucleotides and potentially other short oligoribonucleotides, Orn is also the only known exoribonuclease enzyme that is essential for E. coli, suggesting that removal of the shortest RNAs is an essential cellular function. Organisms naturally lacking the orn gene encode other nanoRNases (nrn) that can complement the conditional E. coli orn mutant. This review covers the history and recent advances in our understanding of these enzymes and their substrates. In particular, we focus on (i) the sources of diribonucleotides; (ii) the discovery of exoribonucleases; (iii) the structural features of Orn, NrnA/NrnB, and NrnC; (iv) the enzymatic activity of these enzymes against diribonucleotides versus other substrates; (v) the known physiological consequences of accumulation of linear dinucleotides; and (vi) outstanding biological questions for diribonucleotides and diribonucleases.


Assuntos
Endorribonucleases , Escherichia coli , Escherichia coli/genética , Transdução de Sinais
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