Table of Contents - Nature Figure 4. Cs+ and Tl+ ions binding sites in the fluoride riboswitch. a,...

a b

c d

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Supplementary Figure 1. ITC-monitored effect of cations on fluoride ion binding to the

fluoride riboswitch. a, ITC binding curve on addition of KF to the fluoride riboswitch in the

presence of 1 mM MgSO4. The Kd is 275 ± 21 μM, the stoichiometry of binding approaches 1:1

(N = 0.75), with estimated thermodynamic parameters of ΔH = -3.3 ± 0.3 Kcal/mol and ΔS = 5.1

cal/mol/deg. b, No binding is detected by ITC on addition of KF to the fluoride riboswitch in the

absence of added MgSO4. c, ITC binding curve on addition of chloride ion (added KCl) to the

fluoride riboswitch in the presence of 20 mM MgSO4. d, No binding is detected by ITC on

addition of fluoride ion (added KF) to the fluoride riboswitch in the presence of 20 mM Li2SO4.

The buffer was 50 mM K-acetate, pH 6.8.


SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature11152

Supplementary Figure 2. Location of four Ir(NH3)63+ binding sites in the ligand-bound

fluoride riboswitch. The refined fluoride riboswitch structure based on a 2.3 Å Ir3+ SAD data set

superposed with the anomalous Ir3+ map (green) coutoured at 3σ level. The four bound Ir3+ ions

are shown as purple spheres and indicated by red arrows. The blue spheres represent symmetry

related Ir3+ ions.



a

b



Supplementary Figure 3. Mn2+ ion binding sites in the fluoride riboswitch. a, The refined

fluoride riboswitch structure (Mn2+ anomalous data) superposed with anomalous map (deep

green) contoured at 3σ level. The bound Mn2+ ions are shown as blue spheres and indicated by

red arrows. b, The three bound Mn2+ ions in the center of the fluoride riboswitch (9σ level) have

been identified in the map. Mn2+ ions replace the three Mg2+ ions coordinated with the bound

fluoride.



a

b



Supplementary Figure 4. Cs+ and Tl+ ions binding sites in the fluoride riboswitch. a, The

refined fluoride riboswitch structure (Cs+ anomalous data) superposed with anomalous map

(deep green) contoured at 3σ level. The bound Cs+ ions are shown as deep purple spheres and

indicated by red arrows. b, The refined fluoride riboswitch structure (Tl+ anomalous data)

superposed with anomalous map (deep green) contoured at 3σ level. The bound Tl+ ions are

shown as brown spheres and indicated by red arrows.



a

b

c



Supplementary Figure 5. Crystal packing interactions in the structure of the ligand-bound

fluoride riboswitch. a, Packing of four fluoride riboswitch molecules, each of which is

represented in a different color. b, Formation of symmetric Watson-Crick G1-C18 pairs between

blue and purple-colored structures of the fluoride riboswitch. Note that 5’-terminal G1 does not

pair with C18 in the same molecule. c, Formation of Watson-Crick G52-C22 pair between

fluoride riboswitches from different tetrameric arrangements of the ligand-bound fluoride

riboswitch. In addition, U51 and U23 from the two molecules are stacked on either side of the

G52-C22 pair. Note that U51 and G52 are 3’-terminal residues.



Supplementary Figure 6. 2Fo-Fc electron density maps for segments of the structure of the

ligand-bound fluoride riboswitch a, 2Fo-Fc electron density map (contoured at 1σ level) for the

structural segment shown in Fig. 2a. b, 2Fo-Fc electron density map (contoured at 1σ level) for

the structural segment shown in Fig. 2b. Mg2+ ions are shown as cyan balls, while a K+ ion is

shown as a gold ball.

a

b



a

b



Supplementary Figure 7. Stereo-views of Fo-Fc omit electron density maps of the fluoride-

binding site and five surrounding nucleotides in the fluoride riboswitch. a, b, Fo-Fc omit

electron density maps contoured at 3σ level (panel a) and at 7σ level (panel b) were calculated

following deletion of fluoride ion, 3 metal ions and bound water molecules. c, d, Fo-Fc omit

electron density maps contoured at 3σ level (panel a) and at 5σ level (panel b) were calculated

following deletion of A6-U7-G8 and U41-G42 segments. Note the inwardly pointing directions

of the five phosphates of these segments.

c

d



a

b

c

d



Supplementary Figure 8. Details of fluoride binding sites in the 1.9 Å fluoride-inhibited

pyrophosphase structure21 (crystals grown from 1 mM MnCl2 and 5 mM NaF-containing

buffer; PDB: 1E6A). The fluoride ion (red) is coordinated by three metal ions, namely two

Mn2+ (purple) ions and one Na+ (palecyan) ion. The three metal ions are coordinated by four

aspartic acid residues, one POP ligand and water molecules. a, An expanded stereo view

highlighting aspartate carboxylate oxygens (in pink, carbon atoms in green) and POP oxygens (in

pink, phosphorus in yellow) directly coordinated to the three metals ions labeled M1 (Na+ in

palecyan), M2 (Mn2+ in purple) and M3 (Mn2+ in purple), that in turn are directly coordinated to

the fluoride ion (in red). b, The fluoride ion is positioned 0.50 Å above the plane formed by the

three metal ions in the structure of the fluoride-inhibited pyrophosphatase. c, A stereo view of

the coordination geometries of fluoride ion (in red) with surrounding metal ions (in purple and

palecyan), and in turn coordination geometries of metals with surrounding aspartate carboxylate

oxygens (in pink) and POP ligand oxygens (in pink) and waters (in silver). Metal coordination

within square-planar positions are shown by dashed blue lines, while coordination in apical

positions are shown by dashed red lines in panels a, c and d. d, The octahedral coordination

geometries around metal ions M1, M2 and M3, together with coordination distances in Å. Note

that the fluoride ion adopts the apical coordination position for metal ions M1, M2 and M3, and

coordination distances to all three metal ions varies between 1.9 and 2.6 Å.

The arrangement of the three metal ions and one fluoride ion in the fluoride-inhibited

pyrophosphate structure is very similar to that observed in the ligand-bound fluoride riboswitch

as shown in Fig. 3.



Supplementary Figure 9. 900 MHz imino proton NMR spectra (15 to 11 ppm) of the T.

petrophila fluoride riboswitch in the free and bound state. a, NMR spectrum of the riboswitch

in the free state (top) and in the fluoride-bound state (bottom). b, NMR spectrum of a mixture of

the free and bound states in slow exchange on the NMR time scale. Spectra were recorded in

buffer containing 50 mM K-acetate-d3, 5 mM Mg-sulfate, 90% H2O/10% D2O, pH 6.8, at 25 oC.



a

b



Supplementary Figure 10. Proposed transcription attenuation mechanism for the T.

petrophila fluoride riboswitch. The complete (sensing domain and expression platform) T.

petrophila fluoride riboswitch could interconvert between two conformations, one in which stem

1 forms in the presence of bound fluoride (ON state) (panel a) and an alternate conformation at

low fluoride concentrations where stem 1 is disrupted as a result of forming a transcription

terminator (OFF state) involving a stable stem loop ending in uracils at the 3’-end (panel b).



Supplementary Table 1. Crystallographic statistics for fluoride riboswitch.

Crystal [Ir(NH3)6]3+

bound Native

Soaked in MnCl2

Soaked in CsCl

Soaked in Tl-acetate

Data collection 24-ID-C 24-ID-E 24-ID-C 24-ID-C 24-ID-C Space group P21212 P21212 P21212 P21212 P21212 Cell dimensions a, b, c (Å) 57.9, 76.5,

42.8 57.0, 75.2, 42.9

57.8, 77.2, 42.1

58.7, 77.1, 42.5

58.2, 77.3, 42.6

α, β, γ (º) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Peak Wavelength 1.1050 0.9792 1.7712 1.7712 1.7712 Resolution (Å) 50.0-2.3

(2.38-2.30) 50.0-2.3 (2.38-2.3)

20.0-2.6 (2.68-2.6)

20.0-2.85 (2.95-2.85)

20.0-2.95 (3.06-2.95)

Rsym or Rmerge 0.058 (0.475)

0.073 (0.622)

0.081 (0.468)

0.106 (0.660)

0.088 (0.444)

I/σI 34.2 (2.3) 31 (2.7) 11.0 (2.8) 13.7 (2.2) 12.9 (2.3) Completeness (%) 99.9 (99.9) 99.4 (99.5) 97.8 (98.9) 99.2 (99.6) 98.3 (99.5) Redundancy 4.5 (4.4) 6.9 (7.0) 3.4 (3.5) 4.4 (4.5) 3.8 (4.0) Unique reflections 16,275 8,726 6,035 4,798 4,257 Refinement Resolution (Å) 46.2-2.3 45.4-2.3 19.8-2.6 20.0-2.85 19.4-2.96 Rwork/Rfree 0.22/0.25 0.22/0.25 0.22/0.26 0.22/0.26 0.23/0.27 No. atoms 1170 1,141 1,139 1,154 1,160 RNA 1122 1,113 1,109 1,122 1109 Fluoride anion 1 1 1 1 1 Cations 12 6 10 12 15 Water 35 21 19 19 35 B-factors RNA 57.3 75.1 69.0 59.6 71.8 Fluoride anion 34.3 37.5 46.2 54.0 67.4 Cations 62.1 63.8 76.2 91.7 91.7 Water 51.3 57.3 56.0 55.0 71.5 R.m.s deviations Bond lengths (Å) 0.03 0.03 0.03 0.04 0.02 Bond angles (º) 0.83 0.78 0.88 1.13 1.23 Values for the highest-resolution shell are in parentheses.



Table of Contents - Nature Figure 4. Cs+ and Tl+ ions binding sites in the fluoride riboswitch. a,...

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