The long and the short of riboswitches

Abstract

Regulatory mRNA elements or riboswitches specifically control the expression of a large number of genes in response to various cellular metabolites. The basis for selectivity of regulation is programmed in the evolutionarily conserved metabolite-sensing regions of riboswitches, which display a plethora of sequence and structural variants. Recent X-ray structures of two distinct SAM riboswitches and the sensing domains of the Mg(2+), lysine, and FMN riboswitches have uncovered novel recognition principles and provided molecular details underlying the exquisite specificity of metabolite binding by RNA. These and earlier structures constitute the majority of widespread riboswitch classes and, together with riboswitch folding studies, improve our understanding of the mechanistic principles involved in riboswitch-mediated gene expression control.

Figure 1

Gene expression control by various riboswitches. Transcriptional attenuation mechanism in the M-box Mg²⁺ sensor (a), lysine riboswitch (b) and flavin mononucleotide (FMN) riboswitch (c). In the absence of ligand, mRNA forms an anti-terminator helix and transcription can proceed through the open reading frame (ORF). In the presence of ligand, the sensing domain binds the cognate metabolite, thereby stabilizing the P1 helix, triggering formation of a transcription terminator in the expression platform and turning off expression of the downstream gene. Complementary anti-terminator sequences are shown in magenta. (d), Regulation of translation initiation by the type III S-adenosylmethionine (SAM) riboswitch. Without ligand, Shine-Dalgarno (SD) sequence is accessible for ribosome entry and translation initiation. In the presence of SAM, SD sequence is paired, thereby preventing ribosome binding.

Figure 2

Tertiary structures of ligand-bound riboswitches and chemical structures of riboswitch ligands. Metabolites are in red. RNA segments in magenta indicate regulatory or ‘switching’ sequences (a–c) that participate in formation of helix P1 or anti-terminator. Grey and blue shading highlights changes in the compounds. (a), M-box Mg²⁺ sensor. Six Mg²⁺ cations are depicted as green spheres. (b), Lysine-sensing domain, the cognate ligand lysine and riboswitch-binding derivatives of lysine. AEC, S-(2-aminoethyl)-L-cysteine; IEL, N6-1-iminoethyl-L-lysine; HArg, L-homoarginine. Potassium cation is in violet. (c), FMN-sensing domain, its cognate ligand FMN, the FMN precursor riboflavin, and antibiotic roseoflavin. (d), Type II S-adenosylmethionine (SAM) riboswitch and the cognate ligand SAM. S-adenosylhomocysteine (SAH) lacks a methyl group (highlighted) and does not bind the riboswitch strongly. SD sequence is indicated in magenta. (e), SAM-III riboswitch.

Figure 3

Ligand recognition by riboswitches. Magenta color depicts nucleotides of the regulatory RNA segment, as in Figure 2. Other nucleotides are colored according with the RNA elements. Hydrogen bonds and hydrogen bond distances are shown with black dashed lines. (a) M-box Mg²⁺ sensor. Mg²⁺ cations Mg1-3 and water molecules are shown as green and red spheres, respectively. Mg²⁺ coordination bonds are depicted by green sticks. (b), FMN-sensing domain. (c), Lysine-sensing domain. K⁺ cation is shown as a violet sphere, while its coordination bonds are depicted by violet sticks. (d), SAM-I riboswitch (top) and schematic representation of SAM recognition (bottom) according with [20]. VDW, van der Waals interactions. Electrostatic interactions are shown with gold dashed lines. (e), SAM-II riboswitch. (f), SAM-III riboswitch.

Figure 4

Folding of purine riboswitches. (a) Crystal structure of the guanine-sensing riboswitch bound to guanine [8]. Discriminatory nucleotide C74 that forms a Watson-Crick base pair with bound guanine is in yellow. In the similar adenine riboswitch, this nucleotide is replaced by uracil. (b) Folding of the guanine riboswitch in the presence of photolabile hypoxanthine derivative [49•]. The three-step folding model based on the experimentally restrained torsion angle molecular dynamics simulations is shown on top. Overlaid structures of the three states simulated according with NMR data, aligned on P2, are shown below. Adapted from Fig. 4a, b in [49•] (copyright 2007 National Academy of Sciences USA). (c) Folding of the adenine-sensing riboswitch domain studied using single-molecule force spectroscopy. Energy landscapes are shown for the folding reactions in the presence (red) and absence (black) of adenine [50••].