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

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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.