A Protonated Cytidine Stabilizes the Ligand‐Binding Pocket in the PreQ 1 Riboswitch in Thermophilic Bacteria

Riboswitches are bacterial mRNA structure elements regulating either transcription or translation of downstream genes in response to high‐affinity binding of a low molecular weight ligand. Among this diverse group of RNA structures, the class‐I pre Q 1 s ensing ribos w itches (QSW) stand out since they are the smallest known natural riboswitches. The preQ 1 sensing riboswitches combine ligand sensing and functional control within a single structural domain that adopts a pseudoknot conformation encapsulating both the cognate ligand and the ribosome binding site. preQ 1 sensing riboswitches also occur in thermophilic bacteria. In these cases, their tertiary structures have to be stable even at temperatures above 60 °C to be functional at the organism's optimal growth temperatures. Despite the available high‐resolution structures of these riboswitches, it is not yet understood which tertiary interactions are primarily responsible for their exceptional temperature stability. Here, we show that an intricate three‐dimensional network of non‐canonical interactions involving various non‐neighboring nucleobases is the origin of the riboswitch's thermostability. An essential part of this network is a so far undetected stably protonated cytidine. It is characterized by an exceptional high pK A value of >9.7 and could be unambiguously identified through the application of modern heteronuclear detected NMR experiments. Thus, the presence or absence of a single proton can modulate the formation of an RNA tertiary structure and ligand binding capacity under extreme environmental conditions.