RESUMEN
RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , ARN sin Sentido/metabolismo , Transcripción Genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.
Asunto(s)
Aptámeros de Nucleótidos/genética , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Técnica SELEX de Producción de Aptámeros , Terminación de la Transcripción Genética , Aptámeros de Nucleótidos/metabolismo , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Nicotinamida-Nucleótido Adenililtransferasa/genética , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Sistemas de Lectura Abierta , Ribosomas/metabolismo , Factores de TiempoRESUMEN
Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor σ(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.
