Knowing the molecular details of the interaction between riboswitch aptamers and their corresponding metabolites is important to understand gene expression. Here we report on a novel in vitro assay to study preQ1 riboswitch aptamers upon binding of 7-Aminomethyl-7-deazaguanine (preQ 1). The assay is based on the ability of the preQ1 aptamer to fold, upon ligand binding, into a pseudoknotted structure that is capable of stimulating -1 ribosomal frameshifting (-1 FS). Aptamers from three different species were found to induce between 7% and 20% of -1 FS in response to increasing preQ1 levels, whereas preQ1 analogues were 100-1000-fold less efficient. In depth mutational analysis of the Fusobacterium nucleatum aptamer recapitulates most of the structural details previously identified for preQ1 aptamers from other bacteria by crystallography and/or NMR spectroscopy. In addition to providing insight into the role of individual nucleotides of the preQ1 riboswitch aptamer in ligand binding, the presented system provides a valuable tool to screen small molecules against bacterial riboswitches in a eukaryotic background.
All Science Journal Classification (ASJC) codes
- Molecular Medicine