Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its target ligand and undergoes conformational changes remain elusive. This gap in understanding has impeded the potential applications of this riboswitch. To bridge this knowledge gap, our study investigated the conformational dynamics of the guanidine-IV riboswitch RNA upon ligand binding. We employed single-molecule fluorescence resonance energy transfer (smFRET) to dissect the behaviors of the aptamer, terminator, and full-length riboswitch. Our findings indicated that the aptamer portion exhibited higher sensitivity to guanidine compared to the terminator and full-length constructs. Additionally, we utilized Position-specific Labelling of RNA (PLOR) combined with smFRET to observe, at the single-nucleotide and single-molecule level, the structural transitions experienced by the guanidine-IV riboswitch during transcription. Notably, we discovered that the influence of guanidine on the riboswitch RNA’s conformations was significantly reduced after the transcription of 88 nucleotides. Furthermore, we proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine, thereby providing insights into its ligand-response mechanism.

The biosynthesis of cyclic 3′,5′-adenosine monophosphate (cAMP) by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here, we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1–7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5–mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR–Gsα signaling regulating the biosynthesis of cAMP.