Natural RNA polymerase aptamers regulate transcription in E. coli [3' RACE]

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 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 growth conditions. The mechanism of iRAPs involves active uncoupling of transcription and translation, making the 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. Overall design: Examination of E. coli 3' ends with two independent replicates sequenced on Illumina HiSeq 2000

为探寻可通过直接结合RNA聚合酶（RNA polymerase, RNAP）调控转录的RNA信号，我们对富集了RNAP结合RNA的大肠杆菌（Escherichia coli, E. coli）基因组文库进行了深度测序。大量天然的RNA聚合酶结合适配体（RNA polymerase-binding aptamers, RAPs）已被定位至大肠杆菌基因组中。超过60%的大肠杆菌基因的mRNA内携带有RAPs。结合体外与体内实验方法，我们对一类可介导依赖Rho（Rho）的转录终止的RAPs亚群进行了系统表征，该亚群被命名为iRAPs。位于必需基因nadD编码区的典型iRAP，可在静止期与氧化应激条件下大幅降低该基因的转录产出，这证明iRAPs可响应生长条件变化调控基因表达。iRAPs的作用机制涉及主动解偶联转录与翻译过程，使新生RNA可被Rho因子结合。反义链编码的iRAPs还可通过降低转录干扰来促进基因表达。简言之，本研究揭示了一类广泛存在的顺式作用RNA信号，它们可全局性调控细菌的转录过程。
整体实验设计：设置两个独立生物学重复，对大肠杆菌的3'端进行检测，测序平台为Illumina HiSeq 2000