Structure and function analysis of a type III preQ1-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence

Abstract

Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine₁ (preQ₁)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ₁, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ₁-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ₁ riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ₁ metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ₁ riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ₁ over the chemically similar metabolic precursor preQ₀. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ₁ riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.

Keywords: Shine-Dalgarno sequence; gene regulation; molecular recognition; preQ(1)-I riboswitch; pseudoknot; translation regulation; x-ray crystallography.

Figure 1

PreQ₁biosynthesis, consensus models of class I preQ₁riboswitches, and representative type I and II cocrystal structures.A, prokaryotic de novo queuosine biosynthetic pathway. Free GTP is first enzymatically converted to preQ₀ by gch1, queE, queD, and queC gene products and is then converted to preQ₁ by queF (10, 65). The yhhQ transporter can salvage both preQ₀ and preQ₁ from the environment in proteobacteria (28). Tgt-derived enzymes insert preQ₁ at position 34 of tRNAs containing GUN anticodons whereupon queA modifies preQ₁ into queuosine (10, 11). B, covariation diagrams of each preQ₁ riboswitch subtype with nucleotides in red, black, and gray indicating 97%, 90%, and 75% sequence conservation (9). Ribbon diagrams of cocrystal structures for the (C) type II Tte (PDB code 6vui) (21) and (D) type I Can (PDB code 8fb3) riboswitches (47). Positions are colored according to pseudoknot pairing and loop regions. PreQ₁ metabolites are depicted as green surface models. preQ1, prequeuosine1.

Figure 2

Length dependence of loop L3 on metabolite binding to the class I type III preQ₁(preQ₁-I_III) riboswitch from E. coli.A, representative isothermal titration calorimetry (ITC) thermogram for preQ₁ binding to the 36-mer WT Eco. Inset: schematic diagram of the L3 loop sequence that was varied in this analysis. K_D, N (stoichiometry), and C values are shown. B, single-deletion ΔG Eco 35-mer variant. C, double deletion ΔGG Eco 34-mer variant. D, triple deletion ΔGGU Eco 33-mer variant. E, Eco 30-mer crystal construct using a linker based on the Tte riboswitch P1-L3 transition. All experiments were performed in duplicate and average thermodynamic parameters are provided in Table 1.

Figure 3

Schematic view, ribbon diagram, and close-up views of the class I type III preQ₁(preQ₁-I_III) riboswitch from E. coli.A, secondary structure of the Eco crystallization construct. Positions are colored according to pseudoknot pairing and loop regions observed in the co-crystal structure; preQ₁ is shown as Q (green). Interactions between specific nucleotides based on the crystal structure are annotated with Leontis-Westhof symbols (66). B, ribbon diagram of the Eco riboswitch based on the co-crystal structure. C, close-up view of the preQ₁-binding pocket floor formed by a quintuple base transition motif comprising two base triples: a planar triple at G5-C15•A25 and a transition triple at C15•A26•U6. D, site I Mn²⁺ ion in stem P1 shown inside anomalous difference Fourier electron density contoured at 6.5σ. PreQ₁ (surface model) binds atop the nearby G5-C15 base pair of stem P1. E, overview of the preQ₁-binding pocket showing preQ₁ interacting with A27 of the SDS (emphasized by yellow-filled nucleotide rings).

Figure 4

Superposition of the Eco type III riboswitch with the type I Can riboswitch and type II Tte riboswitch.A, ribbon diagram of chain A superimposed on all paired atoms of the Eco (purple), Tte (13) (salmon), and Can riboswitches (gold) (26). Close-up views of (B) Eco riboswitch U7 and C8, which produce a tight bend facilitated by the pyrimidine-rich sequence in this region. C, P1-L3 transition showing closest agreement between the Eco riboswitch and the Can riboswitch coordinates. D, α-site preQ₁ binding pocket in Eco, Tte, and Can riboswitches.

Figure 5

Comparison of representative class I riboswitch types at binding pocket ceilings and modes of Shine-Dalgarno sequence sequestration in helix P2.A, close-up top view of Eco (type III) binding pocket ceiling. SDS nucleotide A28 and anti-SDS nucleotides C8 and U11 are highlighted as yellow and cyan base rings; preQ₁ is in green with a semitransparent surface. B, Tte (type II)-binding pocket ceiling (PDB 6vui) (21). C, Can (type I) binding pocket ceiling (PDB 8fb3) (47). D, overview of the Eco riboswitch expression platform. The SDS is sequestered by binding pocket interactions, the pocket ceiling, and helix P2, which engage in canonical and noncanonical interactions with the anti-SDS. The P2 helix of (E) Tte and (F) Can riboswitch with the first two positions of the SDS sequestered by interactions with the anti-SDS.

Figure 6

SPR kinetic analysis of preQ₁and preQ₀metabolite binding to the WT Eco riboswitch.A, representative SPR sensorgrams showing preQ₁ association and disassociation with the WT Eco riboswitch. B, representative SPR sensorgrams showing preQ₀ association and dissociation with the WT Eco riboswitch. C, close-up view of the preQ₁ 7-aminomethyl moiety that donates hydrogen bonds to the nonbridging phosphate oxygen of C12 and O6 of G5. D, hypothetical model of preQ₀ binding to the Eco riboswitch based on superposition of the precursor metabolite on the pyrrolopyrimidine moiety of the experimentally derived preQ₁ model. Kinetic constants are provided in Table 3.

Figure 7

SPR equilibrium binding analysis of Eco preQ₁-I_IIIriboswitch mutants.A, schematic diagrams of the WT Eco 36-mer (left), Eco crystal construct 30-mer (center), and WT Tte 33-mer; preQ₁ is shown in green. Positions colored blue, green, pink, and purple correspond to mutations made for this study that are homologous to those previously made in the Tte riboswitch (13). Positions are colored based on regions of the H-type pseudoknot defined in Figure 3B. Mutants are numbered according to the WT sequence with positions in the crystallization construct shown in parentheses. B, Eco G6DAP/C16U double mutant binding response to preQ₁. C, U7C mutant. D, A33Purine mutant. E, C15U mutant. Binding constants and standard errors are provided in Table 3. All measurements were made in triplicate.

Figure 8

Schematic diagram showing effect of mutations in WT Eco preQ₁-I_IIIriboswitch on switching in a bacterial reporter assay.A, secondary structure of the modified WT Eco 36-mer in a GFPuv reporter construct. Arrows indicate specific mutations to conserved bases. B, preQ₁-dependent dose-response curves of reporter-gene GFPuv normalized fluorescence emission comparing the WT Eco riboswitch with various mutants. E. coli ΔqueC cells containing the riboswitch reporter gene were grown with varying amounts of preQ₁ and fluorescence was measured for each concentration. Curve fits are based on the average of six biological measurements. The negative control lacked the riboswitch and had no functional SDS. C, bar plot showing fold repression of GFPuv fluorescence for WT and mutants; errors are SEM for six biological replicates; ∗∗ p < 0.01 and ∗∗∗ p < 0.001. D, the EC₅₀ fold change of each mutation relative to WT; nd: the mutant EC₅₀ could not be determined. Data from B–D are summarized in Table 4.