Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes - PubMed (original) (raw)

Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes

Arati Ramesh et al. J Mol Biol. 2011.

Abstract

The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 Å by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.

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Figures

Fig. 1

Fig. 1. Metal-dependent compaction of the M-box RNA

(a) Schematic representation of the mechanism for Mg2+-sensing by the M-box riboswitch. Under conditions of low Mg2+ an antiterminator (AT) helix is formed (shown by blue lines) within the mRNA leader region. Under conditions of high Mg2+ the aptamer folds into a conformation that sequesters the left half of the AT and permits formation of a downstream termination site (T). (b) Sequence and secondary structure of the B. subtilis mgtE M-box aptamer RNA used in this study. Circles that are yellow, green, red, and blue correspond to C, U, A, and G, respectively. Dashed lines indicate key tertiary contacts. Numbered green circles denote six Mg2+ observed in the previously published structural model. Circled residues correspond to sites of phosphorothioate interferences _ENREF_31. (c) Analytical ultracentrifugation of M-box aptamer RNA shows compaction of the RNA, represented by a change in the Svedberg coefficient induced by Mg2+ (circles), Mn2+ (triangles) and Ca2+ (squares) in the presence of 100 mM potassium chloride. Compaction of the M-box is achieved at lower concentrations (EC50 = 0.1 mM) of Mn2+ as compared to Mg2+ and Ca2+ (EC50= 0.4 mM). (d-e) SHAPE probing _ENREF_32 of the M-box aptamer upon incubation with increasing divalent metals, shown as a plot of normalized NMIA reactivity versus metal ion concentration. Reactions are shown for (d) physiological (100 mM) and (e) high (2.1 M) concentrations of monovalent ions. Positions that decrease in reactivity with NMIA in the presence of Mg2+ (circles), Mn2+ (triangles) and Ca2+ (squares) are shown. Curve-fitting analysis indicates an EC50 value of 0.41 mM for Mg2+and Ca2+ and 0.12 mM for Mn2+ in the presence of low monovalents. This difference is eliminated in the presence of high concentrations of monovalent ions.

Fig. 2

Fig. 2. Overall structure of Mn2+ bound M-box

(a) Chain A of the two molecules in the asymmetric unit of Mn2+ bound M-box structure (pink) superimposed with Mg2+-bound M-box (blue) shows negligible differences in the backbone and side-chain conformations. Mg2+ ions observed in the Mg2+ bound structure are shown in green. (b) The two molecules in the asymmetric unit of Mn2+-bound M-box are superimposed with chain A in pink and chain X in cyan. (c) Representative portion of the Mn2+ bound M-box structural model is shown (stick representation) with 2Fo-Fc map (mesh) contoured at 1.5σ. (d) Details of interactions observed in the P6 helix are shown. Symmetry related molecules form an inter-molecular kissing loops interaction to stabilize the P6 helix in this crystal form.

Fig. 3

Fig. 3. Locating Mn2+ ions in the M-box structure

(a) The Mn2+ bound M-box structure is shown (grey) with the anomalous difference fourier (mesh) contoured at 7σ (red) and 4σ (blue). (b) Chain A in the Mn2+-bound M-box is shown with potassium ions (purple), previously identified divalent sites (green) and newly identified divalent sites (red). (c) Chain X of the Mn2+ bound M-box is shown with the metals observed in the structural model.

Fig. 4

Fig. 4. Mn2+ ions occupy previously established Mg2+ sites from Core 1 and Core 2 of the M-box

(a) Secondary structure of the B. subtilis mgtE M-box. Red positions denote sites of phosphorothioate interferences. Dashed lines denote key tertiary interactions. Green coloration denotes previously established Mg2+ sites and their inner-sphere interactions. (b) The Mn2+ bound M-box (pink) superimposed with the Mg2+ bound M-box (blue) for the region surrounding the metal sites in Core 1. There are negligible structural differences in the backbone as well as individual nucleotide orientations. Mn2+ ions (green) and metal-coordinating water molecules (black) in the Mn2+ bound M-box are shown along with Mg2+ ions (blue spheres) and metal-coordinating water molecules (magenta) from the Mg2+ bound M-box structures. (c-d) Metal sites M5 and M6 in Core 2 are shown in a color scheme similar to panel A with Mn2+ bound chain A (pink) or chain X (cyan) superimposed on Mg2+ bound M-box (blue). (e) Details of interaction around metal sites 5 (left) and 6 (right) are shown, with distances marked in Angstroms.

Fig. 5

Fig. 5. Mn2+ occupies sites M7 and M9 but not M8 in Core 3

(a-c) Details around metal sites Mn7, putative site M8 and Mn9 are shown with Mn2+ ions as red spheres. Dashed lines mark distances in Å. Key coordinating functional groups are shown along with metal coordinating water molecules (black). (d-f) Surface representation of the corresponding metal sites 7, 8 and 9 in the Mn2+ bound M-box are shown colored by electrostatic potential. Cation binding sites are visible as patches of relatively higher electronegativity.

Fig. 6

Fig. 6. New metal sites that may be functionally relevant

(a) The divalent metal site around Mn10 of chain A of Mn2+-bound M-box structure is shown with Mn2+ ions (red), potassium ions (purple) and water molecules (black). Dashed lines mark distances in Å. (b) Superposition of metal site Mn11 is shown with chain A (pink) and chain X (cyan) of the Mn2+-bound M-box. The site in chain X (cyan) was not occupied by Mn2+, most likely due to differences in backbone conformation as shown herein.

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References

    1. Giedroc DP, Arunkumar AI. Metal sensor proteins: nature's metalloregulated allosteric switches. Dalton Trans. 2007:3107–20. - PubMed
    1. O'Halloran TV. Transition metals in control of gene expression. Science. 1993;261:715–25. - PubMed
    1. Ma Z, Jacobsen FE, Giedroc DP. Coordination chemistry of bacterial metal transport and sensing. Chem Rev. 2009;109:4644–81. - PMC - PubMed
    1. Cayley S, Lewis BA, Guttman HJ, Record MT., Jr. Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo. J Mol Biol. 1991;222:281–300. - PubMed
    1. Froschauer EM, Kolisek M, Dieterich F, Schweigel M, Schweyen RJ. Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica. FEMS Microbiol Lett. 2004;237:49–55. - PubMed

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