Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
mBio. 2020 Sep 15;11(5):e00849-20. doi: 10.1128/mBio.00849-20.
The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In , CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified as having the strongest CsrA-mediated activation across the transcriptome. Here, we determined that CsrA activates expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and coupled transcription-translation assays identified two CsrA binding sites in the leader region of the transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates expression at the posttranscriptional level Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of mRNA. RNA structure prediction revealed an RNA hairpin upstream of the start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of expression may repress biofilm formation under certain conditions. The Csr system of controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5' untranslated leader of mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.
序列特异性 RNA 结合蛋白 CsrA 是保守全局调控 Csr 系统的核心组成部分。在 中，CsrA 调节许多细胞过程，包括生物膜形成、运动性、碳代谢、铁稳态和应激反应。这种调节通常涉及 CsrA 通过与 mRNA 靶标结合来抑制核糖体结合的翻译抑制。虽然 CsrA 还广泛激活基因表达，但尚未证明 CsrA 介导的翻译激活的详细机制。综合转录组研究确定 作为整个 转录组中 CsrA 介导的激活最强的基因。在这里，我们确定 CsrA 在后转录水平激活 表达。凝胶迁移率变动、足迹、印迹和 偶联转录-翻译测定鉴定了 转录本的启动子区域中的两个 CsrA 结合位点，这些位点对于翻译激活至关重要。报告基因融合测定证实 CsrA 在转录后水平激活 表达。此外，两个 CsrA 结合位点中的任一个的结合丧失都消除了 CsrA 依赖性激活。mRNA 半衰期研究表明，CsrA 还促进 mRNA 的稳定性。RNA 结构预测显示，在 起始密码子上游存在一个 RNA 发夹，该发夹可封闭 Shine-Dalgarno (SD) 序列，从而抑制核糖体结合。该发夹还包含两个关键 CsrA 结合位点之一，另一个位点位于上游。我们的结果表明，结合的 CsrA 使 SD 封闭发夹不稳定，从而使核糖体能够结合并启动翻译。由于 YmdA 抑制生物膜形成，因此 CsrA 介导的 表达激活可能会在某些条件下抑制生物膜形成。的 Csr 系统在全球范围内控制基因表达和生理学。该系统的核心组成部分 CsrA 蛋白通过与靶标转录本结合来抑制许多基因的翻译起始，从而与核糖体结合竞争。这种机制的变化非常普遍，以至于 CsrA 有时被称为翻译抑制剂。虽然已经阐明了 CsrA 介导的激活机制，其中结合的 CsrA 通过抑制 RNA 降解来抑制翻译，但尚未定义翻译激活机制。在这里，我们证明 CsrA 结合到 mRNA 5'非翻译启动子中的两个位点通过破坏阻止核糖体结合的结构来激活翻译。CsrA 在激活基因表达中所起的广泛作用表明，类似的激活机制经常发生。
