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 10 3 -, 10 5 -, and 10 6 -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.