Structure-function analysis of cis- and trans-acting RNA thermosensors

结构等于功能；对于RNA来说也是如此。所有细胞RNA的至少某些片段折叠成复杂的结构，对转录、翻译、加工和降解产生影响。为了深入了解人类病原体假结核耶尔森菌的RNA结构，我们采用了两种转录组范围的RNA结构探测方法。在PARS(并行分析的RNA结构)方法中，我们纯化了总RNA，并观察了1.750多个转录本的体外结构特征。为了询问活细菌(在体内)的RNA结构，我们建立了一种名为Lead-Seq的新方法，它利用醋酸铅在不成对的区域破坏磷酸骨架。在不同的温度下应用，这两种方法都提供了关于温度调制的RNA结构的丰富信息。我们对RNA结构特别感兴趣，这种结构在环境温度下阻止核糖体结合，并在宿主体温下融化，从而允许翻译启动。后续研究揭示了一些与毒力相关的RNA温度计(RNAT)的机制细节。利用这些有价值的数据集，我们建议继续我们正在进行的关于RNAs的研究，RNAs控制III型分泌系统和氧化应激反应的基因。一种新的非常有希望的候选基因控制着全球DNA结合蛋白FIS的表达。初步结果表明，FIS在25℃和37℃下相互控制着运动和毒力基因。另一类基于RNA的温度传感器以开关的方式工作，并允许在低温下进行翻译。除了预期的冷休克基因，我们还将分析来自我们的RNA结构资源的几个新的候选基因。这个项目的第二部分将研究一种在很大程度上未被探索的RNA介导的温度控制模式。根据RNA结构学结果，几个小RNA的构象随着温度的变化而变化。我们缩小了两个候选的sRNA，其中mRNA相互作用部位或Hfq结合部位受到影响。我们将研究这些sRNA的信使核糖核酸靶标以及温度对调控的影响。总体而言，这两个工作包都承诺对细菌基因调控中动态RNA结构的分子细节和生理重要性有详细的见解。

Structure equals function; this is also true for RNAs. At least some segments of all cellular RNAs fold into intricate structures that have an impact on transcription, translation, processing and degradation. To provide insights into the RNA structurome of the human pathogen Yersinia pseudotuberculosis, we employed two transcriptome-wide RNA structure probing methods. In the PARS (Parallel Analysis of RNA Structures) approach, we purified total RNA and looked at the in vitro structural features of more than 1.750 transcripts. To interrogate RNA structures in the living bacterium (in vivo), we established a new method called Lead-seq, which takes advantage of lead acetate that breaks the phosphate backbone in unpaired regions. Applied at different temperatures, both approaches provided a wealth of information on the temperature-modulated RNA structurome. We were particularly interested in RNA structures that prevent ribosome binding at environmental temperatures and melt at host body temperature to allow translation initiation. Follow-up studies revealed mechanistic details of a number of such virulence-related RNA thermometers (RNATs). Capitalizing on these valuable datasets, we propose to continue our ongoing studies on RNATs that control genes of the type III secretion system and the oxidative stress response. A new and very promising candidate controls the expression of the global DNA-binding protein Fis. Preliminary results suggest that Fis reciprocally controls motility and virulence genes at 25 and 37°C. Another class of RNA-based thermosensors operates in a switch-like manner and permits translation at low temperatures. Apart from the anticipated cold shock genes, we will analyze several novel candidates derived from our RNA structurome resource. A largely unexplored mode of RNA-mediated temperature control is going to be examined in the second part of this project. According to the RNA structuromics results, several small RNAs change their conformation in response to temperature. We narrowed down two sRNA candidates, in which either the mRNA interaction site or the Hfq-binding site are affected. The mRNA targets of these sRNAs and the impact of temperature on regulation will be examined. Overall, both work packages promise detailed insights into the molecular details and physiological importance of dynamic RNA structures in bacterial gene regulation.