Description:
<jats:title>Abstract</jats:title><jats:p>Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate‐sensing riboswitch in the 5′‐untranslated region of the <jats:italic>E. coli</jats:italic> thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using <jats:sup>31</jats:sup>P NMR spectroscopy. Chemical‐shift changes were observed for the α‐ and <jats:italic>β</jats:italic>‐phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn<jats:sup>2+</jats:sup> ions revealed strong line‐broadening effects for both <jats:sup>31</jats:sup>P signals of the bound TPP; this indicates a Mg<jats:sup>2+</jats:sup> binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion‐mediated manner by the RNA.</jats:p>