The cyclic diguanylate [bis-(3'–5')-cyclic dimeric guanosine monophosphate, c-di-GMP] riboswitch is the first known example of a gene-regulatory RNA that binds to a second messenger. C-di-GMP is widely employed by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The 2-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch employing canonical and non-canonical base pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

The thi-box riboswitch regulates gene expression in response to the intracellular concentration of thiamine pyrophosphate (TPP) in archaea, bacteria, and eukarya. To complement previous biochemical, genetic, and structural studies of this phylogenetically widespread RNA domain, we have characterized its interaction with TPP by isothermal titration calorimetry. This shows that TPP binding is highly dependent on Mg 2+ concentration. The dissociation constant decreases from ;200 nM at 0.5 mM Mg 2+ concentration to ;9 nM at 2.5 mM Mg 2+ concentration. Binding is enthalpically driven, but the unfavorable entropy of binding decreases as Mg 2+ concentration rises, suggesting that divalent cations serve to pre-organize the RNA. Mutagenesis, biochemical analysis, and a new crystal structure of the riboswitch suggest that a critical element that participates in organizing the riboswitch structure is the tertiary interaction formed between the P3 and L5 regions. This tertiary contact is distant from the TPP binding site, but calorimetric analysis reveals that even subtle mutations in L5 can have readily detectable effects on TPP binding. The thermodynamic signatures of these mutations, namely decreased favorable enthalpy of binding and small effects on entropy of binding, are consistent with the P3-L5 association contributing allosterically to TPP-induced compaction of the RNA.

Riboswitches are structured mRNA elements involved in gene regulation that respond to the intracellular concentration of specific small molecules. Binding of their cognate ligand is thought to elicit a global conformational change of the riboswitch, in addition to modulating the fine structure of the binding site. X-ray crystallography has produced detailed descriptions of the three-dimensional structures of the ligand-bound conformations of several riboswitches. We have employed small-angle X-ray scattering (SAXS) to generate low-resolution reconstructions of the ligand-free states of the ligand-binding domains of riboswitches that respond to thiamine pyrophosphate (TPP), and cyclic diguanylate (c-di-GMP), a bacterial second messenger. Comparison of the SAXS reconstructions with the crystal structures of these two riboswitches demonstrates that the RNAs undergo dramatic ligand-induced global conformational changes. However, this is not an universal feature of riboswitches. SAXS analysis of the solution behavior of several other riboswitch ligand-binding domains demonstrates a broad spectrum of conformational switching behaviors, ranging from the unambiguous switching of the TPP and c-di-GMP riboswitches to complete lack of switching for the flavin mononucleotide (FMN) riboswitch. Moreover, the switching behavior varies between examples of the same riboswitch from different organisms. The range of observed behaviors suggests that in response to the evolutionary need for precise genetic regulation, riboswitches may be tuned to function more as dimmers or rheostats than binary on/off switches.