RESUMEN
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.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Interferencia de ARN , ARN sin Sentido/genética , ARN Interferente Pequeño/genética , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Fosforilación , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transformación BacterianaRESUMEN
In Escherichia coli, SdsR and RyeA, a unique pair of mutually cis-encoded small RNAs (sRNAs), act as toxin and antitoxin, respectively. SdsR and RyeA expression are reciprocally regulated; however, how each regulates the synthesis of the other remains unclear. Here, we characterized the biosynthesis of the two sRNAs during growth and investigated their coordinate regulation using sdsR and ryeA promoter mutant strains. We found that RyeA transcription occurred even upon entry of cells into the stationary phase, but its apparent expression was restricted to exponentially growing cells because of its degradation by SdsR. Likewise, the appearance of SdsR was delayed owing to its RyeA-mediated degradation. We also found that the sdsR promoter was primarily responsible for transcription of the downstream pphA gene encoding a phosphatase and that pphA mRNA was synthesized by transcriptional read-through over the sdsR terminator. Transcription from the σ70-dependent ryeA promoter inhibited transcription from the σS-dependent sdsR promoter through transcriptional interference. This transcriptional inhibition also downregulated pphA expression, but RyeA itself did not downregulate pphA expression.
Asunto(s)
Antitoxinas/genética , Toxinas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Antitoxinas/química , Antitoxinas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/metabolismo , Orden Génico , Sitios Genéticos , Regiones Promotoras Genéticas , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , ARN Pequeño no Traducido/genética , Factor sigma/genética , Proteínas Bacterianas/metabolismo , Expresión Génica Ectópica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/metabolismo , Estabilidad del ARN , ARN Pequeño no Traducido/metabolismo , Factor sigma/metabolismoRESUMEN
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.
Asunto(s)
Toxinas Bacterianas/química , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/genética , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Sistemas Toxina-Antitoxina/genética , Proteínas de la Membrana Bacteriana Externa/genética , Toxinas Bacterianas/genética , Muerte Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Escherichia coli/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , ARN Mensajero/genética , Factores de Transcripción/genéticaRESUMEN
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.
