Crystal structures reveal the distinct features of the 2′-dG-III riboswitch in the purine riboswitch family

Abstract Purine riboswitches, located in the 5′-untranslated regions of bacterial messenger RNA, regulate gene expression by sensing purines and their derivatives. A class of the guanine-I riboswitch variants was recently reported to be the third class of 2′-deoxyguanosine (2′-dG) riboswitch termed 2′-dG-III riboswitch. Here we present the crystal structures of the 2′-dG-III riboswitch bound with 2′-dG, guanosine, or guanine. Despite similarities in secondary and overall structures to other purine riboswitches, the 2′-dG-III riboswitch exhibits unique features in its loop–loop interaction, three-way junction, and ligand binding. The 2′-dG-III riboswitch exhibits a tuning fork-like structure with a unique six-tiered interaction within the three-way junction. The second, third, and fourth tiers form the ligand-binding pocket, with a consistent binding mode for the guanine moiety of all three ligands. Structural and biochemical analyses reveal detailed interactions between the 2′-dG-III riboswitch and different ligands, providing insights into its regulatory mechanisms in purine metabolism.