Evidence that the nadA motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD(+)

The nadA motif is a riboswitch candidate present in various Acidobacteria species that was previously identified by bioinformatic analysis of bacterial DNA data sets. More than 100 unique representatives have been identified exclusively upstream of nadA genes, which code for an enzyme in the biosynthetic pathway of the ubiquitous coenzyme NAD(+). The architecture of nadA motif RNAs suggests they use structurally similar tandem ligand-binding aptamer domains to control translation initiation. Biochemical analyses reveal that the first domain selectively binds ligands carrying an adenosine 5′-diphosphate (5′ ADP) moiety, including NAD(+) and its reduced form, NADH. Genetic analyses indicate that a tandem nadA motif RNA suppresses gene expression when NAD(+) is abundant, and that both aptamer domains are required for maximal gene regulation. However, we have not observed selective binding of the nicotinamide moiety of NAD(+) or binding by the second putative aptamer in vitro, despite sequence and structural similarities between the tandem domains.