1. Volume 57, Issue 6, Pages 1088-1098 (March 2015)
Bacterial Riboswitches Cooperatively Bind Ni2+ or Co2+ Ions and Control Expression of Heavy Metal Transporters 
Kazuhiro Furukawa, Arati Ramesh, Zhiyuan Zhou, Zasha Weinberg, Tenaya Vallery, Wade C. Winkler, Ronald R. Breaker 
Molecular Cell 
Volume 57, Issue 6, Pages (March 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2015 57, 1088-1098DOI: (10.1016/j.molcel.2015.02.009)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Structure and Function of czc Motif RNAs
(A) Consensus sequence and structural model for czc motif RNAs. Nucleotides present in greater than 97%, 90%, and 75% of the representatives are depicted as red, black and gray, respectively. Predicted base-paired substructures (P1 through P4), covarying nucleotides (green shading), and non-covarying nucleotides (red shading) are shown.
(B) RNA motif 95 Cbo from Clostridium botulinum. 5′ G residues (lowercase) were added to facilitate in vitro RNA transcription. Disruptive (M1) and restorative (M2) mutations are depicted. Sites of ion-induced changes in spontaneous cleavage are derived from (C) and Figures 2A and S1A.
(C) In-line probing analysis of the 95 Cbo RNA with five divalent metal ions. 5′ 32P-labeled precursor RNAs were subjected to in-line probing without ligand (−) or with 0.3 mM of the divalent ion denoted for each lane. Non-reacted (lane NR) partial digestion of RNA with RNase T1 for cleavage after G residues (lane T1) and alkaline conditions for cleavage at every position (lane OH) are shown. Red brackets highlight cleavage products whose yields are altered by ligand addition.
(D) Periodic table highlighting different effects of metal ions on the czc RNA. The characteristics of various metal cations (20 μM) were established by in-line probing shown in Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 2 Cooperative Ligand Binding of a czc Motif RNA
(A) In-line probing analysis of the 95 Cbo RNA with increasing concentrations of Co2+ or Ni2+ (0.78 μM to 200 μM) shows five regions of structural changes in the RNA (red brackets numbered 1 to 5). Lanes are marked as in Figure 1.
(B) Plot of the normalized fraction of RNA cleavage versus Co2+ derived from the data in (A) shows half-maximal structural change at 6.5 μM Co2+.
(C) Hill plot of the binding data observed for Co2+, Ni2+, and Mn2+. Estimated Hill coefficients (n) are derived from the slopes of the lines. R2 values for the curve analyses are 0.995, 0.995, and for Co2+, Ni2+, and Mn2+, respectively. Also see Figure S2 for Ni2+ and Mn2+ binding assays.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 3 Crystallographic Model of a NiCo in Complex with Co2+ Ions
(A) Secondary structure diagram of Eba NiCo according to the crystallographic model. Seven nucleotides were added at the 5′ and 3′ termini (black) to facilitate crystallization. Two nucleotides in the L4 loop (black) were not modeled due to insufficient electron density. Helices P1-P2 (pink) and P3-P4 (gray) form a “H”-shaped structure stabilized at the junction by four Co2+ ions. Antiterminator nucleotides (cyan) and long-range interactions (dashed) are indicated. Inner sphere (filled symbols) and water mediated (open symbols) contacts to different metals are shown in key.
(B) Crystal structure of NiCo shows two sets of coaxially stacked helices: P1-P2 (pink) and P3-P4 (gray). Interhelical nucleotides coordinate four Co2+ ions (green). Anti-terminator nucleotides 78 to 98 (cyan) are sequestered within P4 and P1, making direct contacts with Co2+ ions. Seven K+ (dark blue) and two Mg2+ (light blue) ions are bound to NiCo.
(C) Nucleotides that undergo modulation by in-line probing (red) lie at or near the Co2+ binding sites.
(D) A Co2+ anomalous difference Fourier map contoured at 4.5σ (yellow mesh) reveals four Co2+ ions (green). C1, C2, and C3 reside on a plane perpendicular to the long axis of NiCo (black disk).
(E and F) Side and top view of the molecule shows a highly splayed out structure with minimum long-range contacts. Also see Figure S3 for in-line probing of Eba NiCo RNA and Table 1 for crystallography data collection and refinement statistics.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Figure 4 Co2+ Binding Sites in a NiCo Aptamer
(A–D) Co2+ ions bind NiCo in octahedral coordination sites. Metal-coordinating nucleotides and water molecules (blue spheres) are indicated.
(E) An experimental SAD electron density map contoured at 2.5σ (blue) corresponding to the NiCo crystallographic model is shown. A cobalt anomalous fourier map contoured at 4.5σ (yellow) helped identify Co2+ ions (green sphere) unambiguously in the structure. Also see Figure S4 for more information on cobalt site electron density.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Figure 5
An Interconnected Network of Molecular Contacts between Co2+ Sites in a NiCo RNA
(A) Co2+ ions (green) bound to NiCo are coordinated by interhelical nucleotides from diametrically opposite sides of the RNA. G87 coordinates cobalt 1 via N7 and cobalt 2 via its ribose oxygen. G45 coordinates cobalt 2 via water mediated contacts with a ribosyl oxygen and cobalt 3 via its N7. These long-range connections (pink dashed lines) extend from A14 to G86 via G45 and G87, connecting three of the Co2+ sites. Coordination distances are listed in Table S1. Also see Figure S5 for global structural changes in a NiCo RNA.
(B) Secondary structure schematic summarizing the connectivity of Co2+ ions with interhelical residues. Co2+ coordinating nucleotides are the most tightly conserved in the NiCo sequence.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

8. Figure 6
Effects of Functional Group Changes on Co2+ Binding by a Bimolecular NiCo Riboswitch Aptamer
(A) The bimolecular aptamer derived from E. bacterium NiCo. Complex formation between RNA Eba 1 (black) and chemically altered RNA Eba 2 (gray) was favored by increasing the base-pairing potential of stems P1 and P4 by the addition of several non-native nucleotides (lowercase letters). Nucleotides G86, G87, and G88 were independently modified as denoted, and circled nucleotides were monitored for spontaneous cleavage using in-line probing assays.
(B) Plot of the KD values for Co2+ binding by the bimolecular aptamer formed from various Eba 2 constructs. Values were estimated from the modulation of in-line probing bands at four different nucleotides as denoted.
(C) Plot of the in-line probing band intensity for various Co2+ concentrations at sites G46 and G40 for the bimolecular aptamer formed using WT Eba 2 RNA or wherein G87 or G88 were replaced with N7-deaza nucleobases.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

9. Figure 7 Control of Gene Expression by a NiCo Riboswitch
(A) Sequence and predicted secondary structure of the E. bacterium NiCo RNA. Nucleotides in P4 and downstream of NiCo (gray shading) can form an altered base paired terminator structure as shown.
(B) In vitro transcription in increasing metal concentrations reveals a Ni2+- and Co2+-dependent increase in antiterminated full-length (FL) RNA transcript and a corresponding decrease in terminated (T) product. M3 mutation that disrupts Co2+ binding exhibits loss of antitermination.
(C) Plot of the yield of full-length transcripts versus Co2+ or Ni2+ concentration for WT and M3 templates. The metal ion concentrations at which elongation efficiency of WT is half-maximal (T50) are indicated with dashed lines. The R2 values for the curve fitting analyses are and for Co2+ or Ni2+ with WT templates, respectively.
(D) Ni2+-induced expression of a gene associated with a NiCo riboswitch in Clostridium scindens. Changes in mRNA level of COG0053 and GAPDH genes in cells exposed to various concentrations of NiCl2 were measured. MDH mRNA levels, which are expected to be unchanged by Ni2+ addition, were used to normalize. Each symbol represents the mean of three independent replicates, and error bars represent SEM. GAPDH, predicted glyceraldehyde-3-phosphate dehydrogenase gene. MDH, predicted malate dehydrogenase gene. Also see Figure S6 for additional in vitro transcription data.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions