Joseph A. Liberman

Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure

Riboswitches are structural elements in the 5′ untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg2+ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Gō-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers′ pre-folded states and their ligand binding affinities.

Riboswitch structure in the ligand-free state

Molecular investigations of riboswitches bound to small‐molecule effectors have produced a wealth of information on how these molecules achieve high affinity and specificity for a target ligand. X‐ray crystal structures have been determined for the ligand‐free state for representatives of the preQ1‐I, S‐adenosylmethionine I, lysine, and glycine aptamer classes. These structures in conjunction with complimentary techniques, such as in‐line probing, NMR spectroscopy, Förster resonance energy transfer, small‐angle scattering, and computational simulations, have demonstrated that riboswitches adopt multiple conformations in the absence of ligand. Despite a number of investigations that support ligand‐dependent folding, mounting evidence suggests that free‐state riboswitches interact with their effectors in the sub‐populations of largely prefolded states as embodied by the principle of conformational selection, which has been documented extensively for protein‐mediated ligand interactions. Fundamental riboswitch investigations of the bound and free states have advanced our understanding of RNA folding, ligand recognition, and how these factors culminate in communication between an aptamer and its expression platform. An understanding of these topics is essential to comprehend riboswitch gene regulation at the molecular level, which has already provided a basis to understand the mechanism of action of natural antimicrobials. WIREs RNA 2012, 3:369–384. doi: 10.1002/wrna.114.

Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during biosynthesis of the essential tRNA base queuosine. We report the first preQ1-II riboswitch structure at 2.3 Å resolution, which uses a novel fold to achieve effector recognition at the confluence of a three-way-helical junction flanking a pseudoknotted ribosome-binding site (RBS). The results account for preQ1-II-riboswitch-mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory RNAs.

A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Significance Riboswitches are RNA molecules found mostly in bacteria that control genes by sensing cellular levels of metabolites, such as the simple organic compound preQ1. The diversity of riboswitches and their potential as novel antibiotic targets continue to elicit interest in these regulatory sequences. Here we present the crystal structure of a newly discovered bacterial preQ1-III riboswitch that senses preQ1 using an unusual, two-part architecture. A complementary analysis of flexibility and dynamics showed that recognition of preQ1 induces riboswitch compaction, while concomitantly enhancing formation of a distant double-helix possessing a regulatory signal that zips and unzips rapidly, producing gene “off” and “on” states. These observations expand our knowledge of riboswitch construction and suggest a broader role for dynamics than previously recognized. PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3′ RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1–P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼0.6 s−1) and undocking (kundock ∼1.1 s−1). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world.

Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.

ITC Analysis of Ligand Binding to PreQ1 Riboswitches

Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, K(A), of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.

Metal Dependence of Ligand Binding and Heavy-Atom Derivatization of Evolutionarily Distinct PreQ1 Riboswitches

Riboswitches are functional RNA elements located most frequently within the 5′-leader sequences of bacterial mRNAs. By directly binding to small molecules via an aptamer domain, a riboswitch can adapt quickly to changes in the concentration of a specific intracellular ligand, thereby establishing a feedback loop that controls gene expression. Here we discuss methods utilized in the structure determination of evolutionarily distinct classes of preQ1 riboswitches known as class I and II, respectively. These riboswitches “sense” the pyrrolopyrimidine metabolite preQ1—an intermediate on the biosynthetic pathway that produces the hypermodified tRNA base queuosine, which imparts translational fidelity. Herein, we describe (1) the use of isothermal titration calorimetry (ITC) to explore metal ion requirements for ligand binding and (2) modifications to crystallization media containing SO 4 2 − or Na+ that were necessary for phase determination using site-bound Os(NH3) 5 3 + or Cs+ ions, respectively. Our experience has shown that simple manipulations to the mother liquor can lead to favorable binding of the latter ions without the need to engineer metal-binding sites, thus making our methods a first-choice approach that is broadly applicable to functional RNAs.