Ribozymes and riboswitches: modulation of RNA function by small molecules

Abstract

Diverse small molecules interact with catalytic RNAs (ribozymes) as substrates and cofactors, and their intracellular concentrations are sensed by gene-regulatory mRNA domains (riboswitches) that modulate transcription, splicing, translation, or RNA stability. Although recognition mechanisms vary from RNA to RNA, structural analyses reveal recurring strategies that arise from the intrinsic properties of RNA such as base pairing and stacking with conjugated heterocycles, and cation-dependent recognition of anionic functional groups. These studies also suggest that, to a first approximation, the magnitude of ligand-induced reorganization of an RNA is inversely proportional to the complexity of the riboswitch or ribozyme. How these small molecule binding-induced changes in RNA lead to alteration in gene expression is less well understood. While different riboswitches have been proposed to be under either kinetic or thermodynamic control, the biochemical and structural mechanisms that give rise to regulatory consequences downstream of small molecule recognition by RNAs mostly remain to be elucidated.

FIGURE 1

Overall structure of representative riboswitch aptamer domains in cartoon form. (A) The TPP riboswitch (21) adopts a Y-shaped structure that recognizes its ligand (red) in an elongated conformation between the pyrimidine-binding helix (cyan) and the pyrophosphate binding helix (green). (B) The preQ₁ riboswitch (11) folds as an H-type pseudoknot, with its ligand continuing the stack between the two A-form helices (cyan and green). (C) The SAM-I riboswitch (17) is organized around a four helix junction (cyan) but also comprises a pseudoknot (green). (D) The glmS ribozyme-riboswitch is comprised of a double-pseudoknot core domain (cyan) and a peripheral domain (green) that consists of a pseudoknot that functions as a 3-helix junction.

FIGURE 2

Ligand recognition by the TPP riboswitch. The pyrimidine is recognized through a combination of base stacking and base pairing. The pyrophosphate is recognized primarily through two chelated divalent cations (M_A2+ and M_B2+) and their hydration sphere.

FIGURE 3

Comparison of base-pairing schemes employed by three riboswitches that recognize guanine and related ligands. The four examples are the (A) purine, (B) preQ₁, and (C) first (g_I) and (D) second guanine (g_II) residues of c-di-GMP bound to its cognate riboswitch.

FIGURE 4

Ligand recognition strategies employed by (A) the Tetrahymena group I intron in recognizing the base of the exogenous guanosine substrate, and (B) flexizyme in positioning the acceptor end (residue 76) of tRNA and phenylalanine.

FIGURE 5

Kinetic vs. thermodynamic control of riboswitches and ribozymes. Transcriptionally controlled riboswitches can operate under kinetic control (green dot) or thermodynamic control (orange dot). Upper panel depicts events relating to riboswitch control as horizontal lines with arrowheads (not to scale). Δt_RNAP represents the time from the completion of aptamer synthesis to the genetic decision point (e.g. RNAP reaching a transcription terminator), shown as a vertical red line; τ represents the time constant for reaching ligand binding and dissociation equilibrium. When Δt_RNAP < τ (τ₂, green), RNAP arrives at the terminator prior to ligand and aptamer reaching binding equilibrium, the riboswitch operates under kinetic control. When Δt_RNAP > τ (τ₁, orange), RNAP arrives at the terminator after ligand and aptamer reach equilibrium and the riboswitch exhibits thermodynamic control. Lower panel depicts a simplified kinetic scheme for competing events occurring at the terminator, depicted as a strong RNA hairpin followed by a slippery U-track. Two yellow ovals depict the RNAP active site. At the terminator, RNAPs are kinetically partitioned into species that read through the terminator or that enter a reversible pause before committing to termination. Nucleotide triphosphates (NTPs) and NusA modulate rates of these events. Additionally, the relative populations of terminator and antiterminator RNAs can be modulated by ligand concentration, ribosomes etc.