Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch

Abstract

Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins. Here we describe a 2.05 angstroms crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B1, an essential participant in many protein-catalysed reactions. Organisms from all three domains of life, including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

Figure 1. Structural models of a TPP riboswitch and its ligands

a, Chemical structures of the natural metabolite TPP and the antimicrobial compound PTPP. b, c, Crystal structure of the TPP-bound sensing domain, showing front (b) and top (c) views. The RNA is in a stick-and-ribbon representation, with bound TPP in red. Stems, loops and junctions are colour coded. d, Schematic depiction of the RNA tertiary fold observed in the structure. Tertiary contacts formed by hydrogen bonds (w, water-mediated bonds) between bases and stacking interactions are represented by thin and thick dashed lines, respectively. Red shading shows nucleotides conserved in more than 97% of sequences (J. E. Barrick and R.R.B., unpublished observations). Encircled M notations represent Mg²⁺ ions.

Figure 2. Structure and interactions in the TPP-binding pocket

a, Stereo view of the central region of the complex containing bound TPP. b, View of TPP, coordinated Mg²⁺ ions (magenta) and water (blue spheres) in the binding pocket. c, Details of the interactions between the HMP ring and RNA. d, Hydrogen bonding between Mg²⁺ ions and RNA.

Figure 3. Tertiary interactions defining TPP riboswitch structure and accessibility to the binding pocket

a, Interaction between J3/2 and P2, mediated by the HMP ring. b, Stabilization of the J2-4 junction by two stacked tetrads (in space-filling representation). c, Interactions between L5 and P3 mediated by three K⁺ ions (red spheres). d, Surface representation of RNA and accessibility to the TPP-binding pocket. TPP is depicted in a stick and mesh representation.

Figure 4. Structural probing of the TPP riboswitch and implications for TPP-mediated gene repression

a, Representative RNase V1 and T2 cleavage patterns for the thiM riboswitch (nt 1–166). 5′ ³²P-labelled RNAs were treated by nucleases in the absence (−) or presence (+) of one, three and ten equivalents of TPP as described in Supplementary Methods and ref. . ⁻OH and Bc stand for ladders prepared by partial digestion with alkali or B. cereus RNase, respectively. The major cleavage protections and enhancements in the presence of TPP are labelled with triangles and stars, respectively. b, Primer extension analysis in the absence and presence of TPP or TMP. RT pauses are indicated by arrows. c, Summary of structure probing experiments. Major TPP protections against V1 (red) and T2 (magenta) RNases are shown. The RT pauses are indicated by arrows. d, Typical mechanisms of TPP-specific gene repression. Top: translation initiation regulation (thiM genes). Bottom: transcription termination regulation (thiC genes). Complementary sequences and alternate base-pairing are shown in blue. SD sequence and initiation codon are shaded green. TPP and Mg²⁺ ions are depicted in red and magenta, respectively. e, Diagram of the OFF state of the E. coli thiM riboswitch.