Dynamic basis of the molecular mechanism of nucleotide secondary messengers-sensing riboswitches by NMR spectroscopy

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The cyclic dinucleotides such as c-di-GMP, c-di-AMP and c-AMP-GMP as well as ZTP are nucleotide secondary messengers that regulate the expression of a multitude of different genes involved for instance in biofilm formation, motility, cell wall biosynthesis, virulence and exoelectrogenesis in bacteria. Regulation of gene expression is based on binding of these nucleotide secondary messengers to riboswitches. Riboswitches are RNA-based regulatory elements in the 5-UTRs of mRNAs capable of regulating transcription or translation. In this project, we will investigate the molecular basis of the riboswitch-mediated gene regulation. In particular, local as well as large scale conformational dynamics are at the heart of riboswitch function. We will investigate these dynamic processes at atomic resolution by applying advanced solution multidimensional heteronuclear NMR-spectroscopy to investigate the functional dynamics of the cyclic dinucleotide and the ZTP-riboswitches. In particular, we will on focus on elucidating the structural changes induced by ligand binding, the functional dynamics of the riboswitches on the level of the aptamer domains, the full-length riboswitches as well as functionally important transcriptional intermediates. Following this approach, we will establish the molecular basis for the dynamic framework of the conformational cross-talk between the aptamer domain and the expression platforms which underlies riboswitch function as well as the molecular basis for their remarkable ligand selectivity. Our project will contribute to a better understanding of the molecular basis for the function of these riboswitches, will uncover molecular strategies to adapt similar molecular functionalities to different environmental conditions and might be helpful to enable the design of riboswitches with novel substrate specifities e. g. for artificial ligand analogues in synthetic biology approaches.