## 4 c-di-AMP Binding Molecules
### c-di-AMP Binding Riboswitches  
Second messenger molecules can bind not only to specific proteins but also to regulatory RNA molecules known as riboswitches [35; 56; 58]. Many classes of riboswitches have been identified bioinformatically based on the presence of conserved RNA motifs within intergenic regions, and have subsequently been shown to bind a variety of molecules including metals, amino acids, carbohydrates and other second messenger molecules in the cells, and thereby regulate downstream genes by pre- or post-transcriptional mechanisms [59]. Riboswitches generally contain a ligand-sensing domain and an expression platform to facilitate the signal transduction of ligand binding and differential expression of the regulated genes [56]. Although several riboswitch classes have been identified based on the presence of RNA motifs, many of them are still considered \"orphan\" as no ligand as yet been identified [70].  
One well-known riboswitch, the _ydaO_ riboswitch, since one of them exists in the promoter region of the _B. subtilis ydaO_ gene, was found to be associated with genes that regulate cell wall metabolism, osmotic stress, and sporulation. However, the riboswitch ligand remained elusive for many years until an unbiased search for the _ydaO_ riboswitch binding molecules utilizing yeast extract as a source of chemically diverse natural metabolites identified AMP as a ligand with weak affinity, and further refinement led to the identification of c-di-AMP as the primary ligand [71; 72]. A mutagenic study of _B. subtilis_ revealed that the _ydaO_ riboswitch is not essential for the bacteria and functions as a transcription terminator in the c-di-AMP bound state. During times of increased c-di-AMP concentration, the riboswitch can act as a genetic off switch to turn off gene expression. The ligand-binding domain of the _ydaO_ riboswitch possesses highly conserved residues for c-di-AMP recognition. It also exhibits a novel platform in the c-di-AMP bound state that includes pseudo-two-fold symmetry of the RNA and a very stable conformation formed after binding to two c-di-AMP molecules [57; 59]. The binding of two c-di-AMP molecules provides a very stable riboswitch conformation, which supports the idea that two c-di-AMP molecules work cooperatively. This conformation reveals that the binding pattern of c-di-AMP to a riboswitch is very different from the binding of c-di-AMP to proteins.  
The _ydaO_ riboswitch is currently the only known c-di-AMP binding riboswitch. Although the riboswitch motif is widely distributed in many bacteria, _ydaO_ is present only in some bacteria that produce c-di-AMP, namely _Bacillus_ and _Clostridium_ species. The _ydaO_ riboswitch has not been found in _Lactococcus_, _Listeria_, _Staphylococcus_, or _Streptococcus_[35]. Interestingly, in _B. subtilis_, the _ydaO_ riboswitch is located within the _ktrAB_ transcript, which encodes a potassium ion transporter (see above), and the _kimA_ (aka ydaO) transcript, which encodes a high affinity potassium ion transporter [73]. The c-di-AMP -riboswitch interaction negatively controls the expression of _kimA_. High levels of extracellular K+ increases the intracellular c-di-AMP concentration, which, in turn, represses the expression of riboswitch controlled genes in the potassium transport system [73].