The structural basis for RNA-mediated gene regulation and for the de novo design of riboswitches: The example of the synthetic neomycin-sensing riboswitch

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The synthetic neomycin-sensing riboswitch is the smallest riboswitch with in vivo activity described to date. The insertion of this 33 nucleotide containing RNA-element into the 5’-untranslated region of an mRNA leads to an efficient down-regulation of gene expression upon binding of the ligand. By combining genetic, biochemical and NMR-spectroscopic techniques we will elucidate the structure and the molecular basis for the regulatory mechanism of this genetic switch. For our studies we will not only use the functional riboswitch RNA. In addition, we found structurally similar RNAs which retained the ability to bind neomycin but are no longer regulatory active as well as aminoglycosides related to neomycin which still bind to the riboswitch with high affinity but where RNA binding does not result in the regulation of gene expression. The comparative analysis of these variants will yield a detailed picture of the molecular determinants of the regulatory activity of this riboswitch as well as starting points for a rational structure based optimization and the de novo design of riboswitches.