Bacterial RNA thermometers: From individual examples to global analyses

结构化RNA区域影响基因表达程序的每一步。许多细菌的热休克和毒力基因都受到RNA温度计（RNAT）的调控，RNA温度计是控制翻译起始的分子拉链。它们采用在低温下阻断核糖体结合位点的结构。温度升高到37°C（毒力基因）或更高（热休克基因）会使结构不稳定并允许翻译。在上一个资助期内，我们对各种天然和合成RNAT进行了分子详细研究。通过使用体外结构探测和下一代测序的组合，我们在三种不同温度下绘制了人类病原体假结核耶尔森氏菌的整个RNA结构组。我们现在想利用这个全面的数据资源，并描述两种不同类型的监管。(i)在对超过25种新型RNAT进行初步表征后，将对其中一些调控元件的结构和生理功能进行评估。我们特别感兴趣的毒力因子CnfY，氧化应激反应和III型分泌系统的组件。(ii)RNA结构组的结果提出了一个监管的概念，其中几个小的监管RNA经历温度依赖性的构象变化，掩盖或揭露其目标相互作用区域。我们将进一步评估这一观察结果，并研究其对靶基因调控的影响。在第三个工作包中，我们计划利用以前开发的体内RNA结构组学方法，目的是了解RNA如何在活细胞中折叠。

Structured RNA regions influence every step in the gene expression program. Many bacterial heat shock and virulence genes are regulated by RNA thermometers (RNATs), which are molecular zippers that control translation initiation. They adopt a structure that blocks the ribosome binding site at low temperatures. A temperature upshift to 37°C (virulence genes) or higher (heat shock genes) destabilizes the structure and permits translation.In the previous funding period, we have studied various natural and synthetic RNATs in molecular detail. By using a combination of in vitro structure probing and next-generation sequencing we mapped the entire RNA structurome of the human pathogen Yersinia pseudotuberculosis at three different temperatures. We now want to take advantage of this comprehensive data resource and characterize two distinct types of regulation. (i) Following the initial characterization of more than 25 novel RNATs, the structure and physiological function of a number of these regulatory elements will be evaluated. We are particularly interested in the virulence factor CnfY, and components of the oxidative stress response and type III secretion system. (ii) The RNA structurome results suggest a regulatory concept, in which several small regulatory RNAs undergo temperature-dependent conformational changes that mask or unmask their target interaction region. We will further evaluate this observation and study its effect on target gene regulation. In a third work package, we plan to capitalize on a previously developed in vivo RNA structuromics approach with the goal to learn how RNAs fold in the living cell.