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 Fe^II. The czcD (NiCo) riboswitch was initially proposed to be specific for Ni^II and Co^II, but we recently showed via a czcD-based fluorescent sensor that Fe^II is also a plausible physiological ligand for this riboswitch class. Here, we provide direct evidence that this riboswitch class responds to Fe^II. Isothermal titration calorimetry studies of the native czcD riboswitches from three organisms show no response to Mn^II, a weak response to Zn^II, and similar dissociation constants (∼1 μM) and conformational responses for Fe^II, Co^II, and Ni^II. Only the iron response is in the physiological concentration regime; the riboswitches' responses to Co^II, Ni^II, and Zn^II 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 Fe^II. 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.