biostudies-literatureChen KKey Realm R&D Program of Guangdong ProvinceNational Natural Science Foundation of ChinaGuangdong Science and Technology DepartmentKey Realm R&D Program of Guangdong ProvinceShenzhen Bay Scholars Programgkaf702https://www.ebi.ac.uk/biostudies/studies/S-EPMC12282948biostudies-literatureUnknown53(14)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.Nucleic acids researchCrystal structures reveal the distinct features of the 2'-dG-III riboswitch in the purine riboswitch family.PMC1228294832400021321711912024A15150125942020B12120300042023B12120600132022B020205000332001639Peng XChen KLiao WRen YLu ZHuang LWang JfalseCrystal structures reveal the distinct features of the 2'-dG-III riboswitch in the purine riboswitch family.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.2025-01-01T00:00:00Z2025 Jul2026-07-01T03:15:36.16Z2025-08-12T03:05:02.562ZS-EPMC122829484069484710.1093/nar/gkaf702