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Recognition of the bacterial alarmone ZMP through long-distance association of two RNA subdomains

Nature Structural & Molecular Biology volume 22, pages 679–685 (2015)Cite this article

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Abstract

The bacterial alarmone 5-aminoimidazole-4-carboxamide riboside 5′-triphosphate (AICAR triphosphate or ZTP), derived from the monophosphorylated purine precursor ZMP, accumulates during folate starvation. ZTP regulates genes involved in purine and folate metabolism through a cognate riboswitch. The linker connecting this riboswitch's two subdomains varies in length by over 100 nucleotides. We report the cocrystal structure of the Fusobacterium ulcerans riboswitch bound to ZMP, which spans the two subdomains whose interface also comprises a pseudoknot and ribose zipper. The riboswitch recognizes the carboxamide oxygen of ZMP through an unprecedented inner-sphere coordination with a Mg2+ ion. We show that the affinity of the riboswitch for ZMP is modulated by the linker length. Notably, ZMP can simultaneously bind to the two subdomains even when they are synthesized as separate RNAs. The ZTP riboswitch demonstrates how specific small-molecule binding can drive association of distant noncoding-RNA domains to regulate gene expression.

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Figure 1: Structure of the F. ulcerans ZTP riboswitch.
Figure 2: Critical ligand-RNA and RNA-RNA interactions in the ZTP riboswitch.
Figure 3: Effect of linker length on ZMP binding affinity.
Figure 4: Transcription antitermination by ZTP riboswitch linker variants.
Figure 5: Ligand recognition by purine and purine-like riboswitches.

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Acknowledgements

We thank the staff at beamlines 5.0.1 and 5.0.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory for crystallographic data collection; R. Trachman for SAXS data collection; G. Piszczek (US National Heart, Lung, and Blood Institute, NHLBI, National Institutes of Health (NIH)) for isothermal titration calorimetry support; and L. Fang, S. Seifert and X. Zuo at beamline 12-ID-C of the Advanced Photon Source, Argonne National Laboratory (ANL) for SAXS support. SAXS data were collected in a core facility of the Center for Cancer Research, US National Cancer Institute (NCI) allocated under agreement between NCI and ANL (PUP-24152). We also thank S. Bachas, M. Chen, C. Fagan, M. Lau, R. Trachman, K. Warner and J. Zhang for discussions. This work was partly conducted at the ALS, on the Berkeley Center for Structural Biology beamlines, which are supported by the NIH. Use of ALS and APS was supported by the US Department of Energy. This work was supported in part by the intramural program of the NHLBI, NIH, and by a Lenfant Biomedical Fellowship to C.P.J.

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  1. Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    Christopher P Jones & Adrian R Ferré-D'Amaré

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Contributions

C.P.J. designed and carried out experiments, data analysis, diffraction data collection and structure determination. C.P.J. and A.R.F.-D. prepared the manuscript.

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Correspondence to Adrian R Ferré-D'Amaré.

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Integrated supplementary information

Supplementary Figure 1 Predicted secondary structures of the ZTP riboswitches.

(a) Halothermothrix orenii, (b) Klebsiella pneumoniae, (c) Paenibacillus sp. HGF5, (d) Spirochaeta thermophila, (e) Thermobispora bispora, (f) Thermosinus carboxydivorans, (g) Thermobacillus composti, and (h) F. ulcerans + 102-nt linker from environmental sample 3278. Secondary structures are based on the F. ulcerans secondary structure as seen in the crystal structure. Additional helices predicted to form at the 5´ end of the RNA are labeled “P0”, and the insertion domain helix of T. bispora is labeled “P5”.

Supplementary Figure 2 Crystallization of F. ulcerans ZTP riboswitch.

(a) Image of ZTP riboswitch crystals growing from a polyethylene glycol precipitate, as described in methods. Bar denotes 100 μm. (b) Denaturing polyacrylamide gel of a single riboswitch crystal. Lanes 1–3: riboswitch RNA control: 2.5, 1.25, and 0.625 μg purified RNA. Lanes 4–6: sequential crystal wash solutions. Lane 7: riboswitch crystal. (c) Density-modified 2|Fo|-|Fc| SAD map (blue mesh) used for initial model building, contoured at 2 σ.

Supplementary Figure 3 Conservation of ZTP riboswitch and mapping onto the F. ulcerans riboswitch.

(a) Conservation/covariation data published by Breaker and coworkers (Kim, P. B. et al, Mol Cell, 57, 317-28, 2015) mapped onto the sequence and secondary structure of the F. ulcerans riboswitch. Red nucleotides are more than 97% conserved, blue nucleotides are more than 90% conserved, and gray nucleotides are more than 75% conserved. (b) Cartoon view of the same conservation data mapped onto the crystallographic model. Coloring is the same as in A, except black residues (not conserved) are shown in white.

Supplementary Figure 4 Small-angle X-ray scattering (SAXS) analysis of ZTP riboswitches.

(a) Size-exclusion chromatography traces for the F. ulcerans, H. orenii, and T. carboxydivorans pfl RNAs. (b) Denaturing and native polyacrylamide gels of pooled and concentrated monomer fractions, visualized by staining with ethidium bromide. Lane 1: H. orenii. Lane 2: F. ulcerans. Lane 3: T. carboxydivorans. (c) SAXS data for the F. ulcerans, H. orenii, and T. carboxydivorans RNAs in the presence (red) and absence (black) of ZMP. Arrow indicates the presence of aggregation in the T. carboxydivorans samples without ZMP. (d) Kratky plot for the F. ulcerans riboswitch in the presence (black) and absence (red) of ZMP. (e) Cartoon view of the crystal contact interface formed between two RNA dimers (left) and overall view of the tetramer in the crystal (right). (f) Normalized size-exclusion chromatography traces of purified monomeric (left) and dimeric (right) fractions of the F. ulcerans riboswitch prior to isothermal titration calorimetry (ITC) measurements (black) and after ITC measurements (red).

Supplementary Figure 5 Representative isothermal calorimetry titration experiments and single-round transcription experiments of F. ulcerans ZTP riboswitch linker variant RNAs.

(a) 20 μM wild-type ZTP riboswitch titrated with 200 μM ZMP. (b) 50 μM +10 A linker variant titrated with 1 mM ZMP. (c) 50 μM +20 A linker variant titrated with 1 mM ZMP. (d) 50 μM +env3278 linker variant titrated with 1 mM ZMP. (e) 40 μM ZTP riboswitch lacking the pseudoknot (Δ55-75) with 40 μM 59–75 added in trans titrated with 800 μM ZMP. (f) 40 μM ZTP riboswitch Δ55-75 titrated with 800 μM ZMP. (g) Representative transcription termination experiment of F. ulcerans ZTP riboswitch linker variants. Lanes 1–3: wild-type template in the presence of 0, 100, and 1000 μM ZMP. Lanes 4–6: +10A linker template in the presence of 0, 100, and 1000 μM ZMP. Lanes 7–9: +20A linker template in the presence of 0, 100, and 1000 μM ZMP. Lanes 10–12: +env3268 linker template in the presence of 0, 100, and 1000 μM ZMP. Bands corresponding to full-length (F) and terminated (T) transcription products are indicated.

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Jones, C., Ferré-D'Amaré, A. Recognition of the bacterial alarmone ZMP through long-distance association of two RNA subdomains. Nat Struct Mol Biol 22, 679–685 (2015). https://doi.org/10.1038/nsmb.3073

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