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

结构等于功能;对于RNA也是如此。所有细胞RNA中至少有一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结构组，我们采用了两种转录组范围的RNA结构探测方法。在PARS（RNA结构的平行分析）方法中，我们纯化了总RNA，并观察了超过1.750个转录本的体外结构特征。为了在活细菌（体内）中询问RNA结构，我们建立了一种名为Lead-seq的新方法，该方法利用醋酸铅破坏未配对区域的磷酸骨架。在不同的温度下应用，这两种方法提供了丰富的信息的温度调制的RNA结构。我们特别感兴趣的是RNA结构，它可以在环境温度下阻止核糖体结合，并在宿主体温下融化以允许翻译起始。后续研究揭示了许多这种与毒性相关的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控制III型分泌系统和氧化应激反应基因的RNAT的研究。一个新的和非常有前途的候选人控制全球DNA结合蛋白Fis的表达。初步结果表明，Fis factors在25和37 ° C下控制运动和毒力基因。另一类基于RNA的热传感器以类似开关的方式工作，并允许在低温下翻译。除了预期的冷休克基因，我们将分析几个新的候选人来自我们的RNA结构组资源。在本项目的第二部分，将研究一种基本上未被探索的RNA介导的温度控制模式。根据RNA结构组学结果，一些小RNA响应于温度而改变其构象。我们缩小了两个sRNA候选者，其中mRNA相互作用位点或Hfq结合位点受到影响。这些sRNA的mRNA靶点以及温度对调控的影响将被检查。总的来说，这两个工作包承诺详细了解细菌基因调控中动态RNA结构的分子细节和生理重要性。

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.