RESUMO
Most small noncoding RNAs (sRNAs) are known to base pair with target mRNAs and regulate mRNA stability or translation to trigger various changes in the cell metabolism of Escherichia coli. The SdsR sRNA is expressed specifically during the stationary phase and represses tolC and mutS expression. However, it was not previously known whether the growth-phase-dependent regulation of SdsR is important for cell growth. Here, we ectopically expressed SdsR during the exponential phase and examined cell growth and survival. We found that ectopic expression of SdsR led to a significant and Hfq-dependent cell death with accompanying cell filamentation. This SdsR-driven cell death was alleviated by overexpression of RyeA, an sRNA transcribed on the opposite DNA strand, suggesting that SdsR/RyeA is a novel type of toxin-antitoxin (T/A) system in which both the toxin and the antitoxin are sRNAs. We defined the minimal region required for the SdsR-driven cell death. We also performed RNA-seq analysis and identified 209 genes whose expression levels were altered by more than two-fold following pulse expression of ectopic SdsR at exponential phase. Finally, we found that that the observed SdsR-driven cell death was mainly caused by the SdsR-mediated repression of yhcB, which encodes an inner membrane protein.
Assuntos
Toxinas Bacterianas/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Sistemas Toxina-Antitoxina/genética , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , Morte Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA. Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Interferência de RNA , RNA Antissenso/genética , RNA Interferente Pequeno/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Fosforilação , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transformação BacterianaRESUMO
Many small RNAs (sRNAs) regulate gene expression by base pairing to their target messenger RNAs (mRNAs) with the help of Hfq in Escherichia coli. The sRNA DsrA activates translation of the rpoS mRNA in an Hfq-dependent manner, but this activation ability was found to partially bypass Hfq when DsrA is overproduced. The precise mechanism by which DsrA bypasses Hfq is unknown. In this study, we constructed strains lacking all three rpoS-activating sRNAs (i.e., ArcZ, DsrA, and RprA) in hfq+ and Hfq- backgrounds, and then artificially regulated the cellular DsrA concentration in these strains by controlling its ectopic expression. We then examined how the expression level of rpoS was altered by a change in the concentration of DsrA. We found that the translation and stability of the rpoS mRNA are both enhanced by physiological concentrations of DsrA regardless of Hfq, but that depletion of Hfq causes a rapid degradation of DsrA and thereby decreases rpoS mRNA stability. These results suggest that the observed Hfq dependency of DsrA-mediated rpoS activation mainly results from the destabilization of DsrA in the absence of Hfq, and that DsrA itself contributes to the translational activation and stability of the rpoS mRNA in an Hfq-independent manner.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Pequeno RNA não Traduzido/genética , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Expressão Ectópica do Gene , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA , Pequeno RNA não Traduzido/metabolismo , Fator sigma/metabolismoRESUMO
Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA. Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.
Assuntos
Escherichia coli/genética , Inativação Gênica , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Conformação de Ácido NucleicoRESUMO
Bacterial small RNAs (sRNAs) are known regulators in many physiological processes. In Escherichia coli, a large number of sRNAs have been predicted, among which only about a hundred are experimentally validated. Despite considerable research, the majority of their functions remain uncovered. Therefore, collective analysis of the roles of sRNAs in specific cellular processes may provide an effective approach to identify their functions. Here, we constructed a collection of plasmids overexpressing 99 individual sRNAs, and analyzed their effects on biofilm formation and related phenotypes. Thirty-three sRNAs significantly affecting these cellular processes were identified. No consistent correlations were observed, except that all five sRNAs suppressing type I fimbriae inhibited biofilm formation. Interestingly, IS118, yet to be characterized, suppressed all the processes. Our data not only reveal potentially critical functions of individual sRNAs in biofilm formation and other phenotypes but also highlight the unexpected complexity of sRNA-mediated metabolic pathways leading to these processes.
Assuntos
Biofilmes , Escherichia coli/genética , Fímbrias Bacterianas/genética , RNA Bacteriano/metabolismo , Northern Blotting , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/genéticaRESUMO
Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.