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Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch

Abstract

The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches1,2,3. Flavin mononucleotide (FMN)-specific riboswitches4,5, also known as RFN elements6, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin7. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg2+-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

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Figure 1: Overall structure and tertiary interactions of the FMN-bound F. nucleatum riboswitch.
Figure 2: Recognition of FMN by its riboswitch.
Figure 3: Interactions of FMN analogues with the riboswitch.
Figure 4: Probing the FMN riboswitch tertiary structure.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates of the X-ray structures of the FMN riboswitch are deposited in the RCSB Protein Data Bank under the following accession numbers: bound to FMN, transcribed RNA, 3F2Q; bound to FMN, two-strand composition, 3F4E; bound to riboflavin, 3F4G; bound to roseoflavin, 3F4H; [Ir(NH3)6]3+-soaked, 3F2T; Cs+-soaked 3F2X; Ba2+-soaked, 3F2W; Mn2+-soaked, 3F2Y; [Co(NH3)6]3+-soaked, 3F30.

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Acknowledgements

We thank the personnel of beamline X29 at the Brookhaven National Laboratory and beamline 24-ID-C at the Advanced Photon Source, Argonne National Laboratory, funded by the US Department of Energy. We thank L. Jaeger (University of California, Santa Barbara) for discussions and O. Ouerfelli (Memorial Sloan-Kettering Cancer Center, New York) for the synthesis of iridium hexamine. D.J.P. was supported by funds from the National Institutes of Health.

Author Contributions L.H. crystallized the F. nucleatum FMN riboswitch and performed binding experiments, A.S. determined the structures and performed footprinting experiments, and A.S. and D.J.P. wrote the manuscript with the assistance of L.H. All authors discussed the results and commented on the manuscript.

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Correspondence to Alexander Serganov or Dinshaw J. Patel.

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This file contains Supplementary Figures 1-19 with Legends, Supplementary Tables 1-2 and Supplementary References. This file was replaced on February 4th, 2009 to correct 2 chemical formulae. (PDF 9097 kb)

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Serganov, A., Huang, L. & Patel, D. Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch. Nature 458, 233–237 (2009). https://doi.org/10.1038/nature07642

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