Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - RiboDisc (Discovery of novel orphan riboswitch ligands)

Teaser

Riboswitches are mRNA-based gene-regulatory elements triggered by direct interactions with small molecular ligands. They are exciting targets for novel antibiotic strategies. Many putative riboswitches have been identified using bioinformatics. However, ligand identification...

Summary

Riboswitches are mRNA-based gene-regulatory elements triggered by direct interactions with small molecular ligands. They are exciting targets for novel antibiotic strategies. Many putative riboswitches have been identified using bioinformatics. However, ligand identification is getting more complicated since many motifs are not expected to be involved in simple feedback regulation mechanisms. For such “classical” riboswitches ligands have been assigned in the past based on testing metabolites selected by educated guesses guided by the associated gene contexts. We are convinced that this approach limits the identification of riboswitches that play regulatory roles in more complex bacterial processes such as virulence, detoxification, communication, and life style adaptations. Within this project, new riboswitch classes will be identified and characterized, paving the way for the development of antibiotics with novel modes of action. We will establish a systematic, robust and unbiased approach for identifying intracellular RNA ligands by fishing small molecules from lysates as well as screening fractionated cellular extracts. The methodology shows great potential for assigning novel riboswitch classes. The proposed research is highly relevant for one of the major biomedical challenges of the coming decades: Since riboswitches are effective antibacterial targets, the identification of novel riboswitch / ligand interactions has immediate implications for establishing future antibiotic strategies necessary to keep in check the progressing problem of bacterial drug resistance.

Work performed

The project has started successfully by establishing the proposed methods for preparing and screening bacterial small molecule extracts for novel orphan riboswitch ligands. Ligand identification is ongoing for several orphan riboswitch motifs. In a first publication in JACS (DOI: 10.1021/jacs.7b06141) we have examined variants of riboswitches that bind to bacterial signaling molecules. For this purpose, we synthesized novel cyclic dinucleotides and assayed them for binding to described c-di-AMP, c-di-GMP and c-AMP-GMP riboswitches. In addition, we investigated their immunostimulatory properties. While we studied certain RNA motifs associated with genes of the central carbon and energy metabolism, we discovered the first lysine degradation pathway in E. coli. It appears wide-spread in bacteria and respresents the first glucogenic pathway of lysine degradation. The results are published in Nature Communications (DOI: 10.1038/s41467-018-07563-6).

Final results

We expect to discover several new orphan riboswitch ligands. In addition, we will gain novel insights into the physiology, microbiology, and biochemistry of RNA-regulated activities and pathways in bacteria.