The czcD (NiCo) Riboswitch Responds to Iron(II)

Abstract

Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to FeII. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to NiII and CoII but thought not to respond to FeII, on the basis of aerobic, in vitro assays; its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to FeII, as well as to MnII, CoII, NiII, and ZnII, but only in the cases of FeII and MnII do the apparent K d values (0.4 and 11 μM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensorwhich is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for FeIIresponds to iron in Escherichia coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for czcD riboswitches, although a response to general metal stress cannot be ruled out at present.

Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET

“…4 B) which fit to a binding isotherm with a Hill coefficient of 1.9 and an EC 50 = 0.84 ± 0.05 µM. These data are consistent with previous results from in-line probing analysis ( Furukawa et al 2015 ) and a chimeric Spinach2 sensor assay ( Xu and Cotruvo 2020 ). The E' FRET value measured in the absence of Co 2+ was 0.09, likely resulting from intrinsic flexibility of the four-way junction in the absence of Co 2+ .…”

Sequential Folding of the Nickel/Cobalt Riboswitch Is Facilitated by a Conformational Intermediate: Insights from Single-Molecule Kinetics and Thermodynamics

“…65,66 A cooperative Co 2+ response of the NiCo riboswitch may therefore be advantageous by allowing cellular [Co 2+ ] to be maintained near K d but where high cytotoxicity requires any additional Co 2+ to be immediately removed. 6,7 Note that while the present K d values are in quantitative agreement with the recent studies of NiCoriboswitch-based fluorescent sensors titrated in citrate buffer, 67 they are roughly an order of magnitude lower (i.e., more sensitive) than previous in-line probing results. 16 We do not know the reason for these differences but can speculate that the presence of high [Mg 2+ ] in the in-line probing studies may interfere with ligand recognition/binding.…”

“…Specifically, the increase in k I→F at low [Ni 2+ ] indicates a lower cooperativity ( n = 1.8(10)) for Ni 2+ -promoted folding, yet which is compensated by an even tighter ligand affinity ( K d = 0.26(7) μM) (Table ). The results suggest that the NiCo riboswitch has evolved to trigger at lower levels of cognate ligand but more gradually toward removal of excess Ni 2+ . A second difference in the NiCo riboswitch behavior can be seen in the saturation value of k I→F , which is nearly 3 times lower for Ni 2+ than Co 2+ and therefore predicts only ∼50% of the folded population under saturating [Ni 2+ ] conditions.…”

Coordination Complexes to Combat Bacterial Infections: Recent Developments, Current Directions and Future Opportunities

“…discovered iron‐sensing riboswitches. These riboswitches present within the coding regions of messenger RNAs (mRNAs), bind to Fe 2+ in the presence of other metal ions with high specificity and undergo conformational changes enabling a genetic response increasing the translation of its associated mRNA in bacteria [56, 57] …”