Reconsidering the czcD (NiCo) Riboswitch as an Iron Riboswitch

Recent work has proposed a new mechanism of bacterial iron regulation: riboswitches that undergo a conformational change in response to FeII. The czcD (NiCo) riboswitch was initially proposed to be specific for NiII and CoII, but we recently showed via a czcD-based fluorescent sensor that FeII is also a plausible physiological ligand for this riboswitch class. Here, we provide direct evidence that this riboswitch class responds to FeII. Isothermal titration calorimetry studies of the native czcD riboswitches from three organisms show no response to MnII, a weak response to ZnII, and similar dissociation constants (∼1 μM) and conformational responses for FeII, CoII, and NiII. Only the iron response is in the physiological concentration regime; the riboswitches' responses to CoII, NiII, and ZnII require 103-, 105-, and 106-fold higher "free"metal ion concentrations, respectively, than the typical availability of those metal ions in cells. By contrast, the "Sensei"RNA, recently claimed to be an iron-specific riboswitch, exhibits no response to FeII. Our results demonstrate that iron responsiveness is a conserved property of czcD riboswitches and clarify that this is the only family of iron-responsive riboswitch identified to date, setting the stage for characterization of their physiological function.