Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch

Abstract

Riboswitches are structured RNA elements that bind a specific ligand to control expression of the gene to which they are linked. Several bacterial genes involved in the transport and synthesis of riboflavin and related compounds are regulated by a riboswitch that binds flavin mononucleotide (FMN). Serganov et al. report the unusual structure of the metabolite-sensing domain bound to FMN, riboflavin and an antibiotic. The relatively open ligand-binding pocket suggests that antimicrobials based on FMN could be devised. This paper reports the unusual structure of the metabolite-sensing domain of a flavin mononucleotide (FMN)-specific riboswitch bound to FMN, riboflavin and an antibiotic. The relatively open ligand-binding pocket suggests that antimicrobials based on FMN could be devised. 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.