Structure and mechanism of purine-binding riboswitches

Abstract

A riboswitch is a non-protein coding sequence capable of directly binding a small molecule effector without the assistance of accessory proteins to regulate expression of the mRNA in which it is embedded. Currently, over 20 different classes of riboswitches have been validated in bacteria with the promise of many more to come, making them an important means of regulating the genome in the bacterial kingdom. Strikingly, half of the known riboswitches recognize effector compounds that contain a purine or related moiety. In the last decade, significant progress has been made to determine how riboswitches specifically recognize these compounds against the background of many other similar cellular metabolites and transduce this signal into a regulatory response. Of the known riboswitches, the purine family containing guanine, adenine and 2'-deoxyguanosine-binding classes are the most extensively studied, serving as a simple and useful paradigm for understanding how these regulatory RNAs function. This review provides a comprehensive summary of the current state of knowledge regarding the structure and mechanism of these riboswitches, as well as insights into how they might be exploited as therapeutic targets and novel biosensors.

Figure 1

A typical riboswitch that regulates transcription. (top) Binding of the effector ligand binding to the aptamer domain (AD, green) fates the expression platform (EP, yellow) to form a rho-independent transcriptional terminator. This stem-loop structure causes RNAP to disengage from mRNA synthesis, thereby turning off expression. (bottom) In the absence of ligand binding to the aptamer, the P1 helix is destabilized and the “switching sequence” is available to form the competing antiterminator structure disrupts formation of the terminator as well, allowing for full transcription of the mRNA.

Figure 2

Structure of the guanine riboswitch. (a) Secondary structure of the guanine riboswitch. Only nucleotide positions whose identity is >97% conserved across all phylogenetic variants are presented; all other nucleotide positions are denoted as dark circles. Canonical Watson-Crick base pairing interactions are shown by dashes, noncanonical pairs are denoted by open circles, and the backbone trace is given through the grey dashed line. Paired regions are denoted by “P” (P1 – P3 in this case), terminal hairpin loops by “L” (L2 and L3), and joining regions by “J” (J2/3). This convention will be used throughout all the figures in this review. Regions of interest are shaded: the binding pocket is colored green, the specificity pyrimidine is cyan, and the two base quadruples that establish the L2–L3 interaction is orange. (b) Tertiary structure of the guanine riboswitch aptamer domain bound to guanine (PDB ID 1Y27). The shaded regions reflect those in part (a). (c) Guanine recognition is effected through a base triple with two universally conserved pyrimidine residues (U51 and C74). The specificity pyrimidine is shown in blue (74) and is a uridine in all adenine responsive variants. The dots, representing the van der Waals surface of the atoms highlight that the nucleobase is almost entirely surrounded by RNA.

Figure 3

Recognition of 2’-deoxyguanosine by the purine (PDB ID 3DS7). The nucleobase moiety hydrogen bonds to C51 and C74; note that C51 is shifted towards C74 relative to the guanine riboswitch to provide room for the 2’-deoxyribose moiety. To further accommodate the sugar, A47 is shifted away from C51 (compare to Fig. 2c). Further interactions with the sugar are made with the adjacent base triple in P1.

Figure 4

Structure of the preQ₁-I riboswitch aptamer. (a) Secondary structure of the aptamer domain. The binding site for preQ₁ (magenta “Q”) is shaded in green. (b) Tertiary structure of the Thermoanaerobacter tengcongensis preQ₁ aptamer in complex with Q₀ (PDB ID 3GCA). (c) Recognition of Q₀ by the T. tengcongensis aptamer. Note a hydrogen bonding pattern very similar to that of the purine riboswitch (Fig. 2c). (d) Recognition of Q₁ by the B. subtilis preQ₁ riboswitch aptamer domain (type II; PDB ID 3FU2), emphasizing additional hydrogen bonding interactions that give rise to the riboswitch’s preference for Q₁ over Q₀ and guanine.

Figure 5

Structure of the class I cyclic-di-GMP riboswitch aptamer. (a) Secondary structure of the aptamer domain. The binding site is shaded green, the tetraloop-tetraloop receptor (TL-TLR) is shown in orange, and a second conserved set of tertiary interactions that serve to anchor P2 and P3 together in red. (b) Tertiary structure of the ligand-aptamer complex (PDB ID 3IRW). (c) Recognition of c-di-GMP by the three way junction of the aptamer, emphasizing the hydrogen bonding network between the ligand and RNA. (d) Another view of the recognition of Gβ of c-di-GMP by the aptamer.

Figure 6

Structure of the class II cyclic-di-GMP riboswitch aptamer. (a) Secondary structure of the aptamer. The binding site is shaded in green, the pseudoknot in cyan, and the kink-turn (KT) in yellow. (b) Tertiary structure of the aptamer (PDB ID 3Q3Z). (c) Recognition of cyclic-di-GMP by the riboswitch. Note that the mode of recognition of both G-α and G-β is very different from the class I riboswitch, but the in both cases an adenosine intercalates between the guanosine residues.

Figure 7

Structure of the tetrahydrofolate (THF) riboswitch aptamer. (a) Secondary structure of the THF riboswitch aptamer domain, highlighting the two separate THF binding sites (green), the three-way junction (3WJ, orange), and the pseudoknot (PK, red). Note that only a few nucleotides are universally conserved in this aptamer, none of which directly contact the ligands. (b) Tertiary structure of the THF aptamer (PDB ID 3SD1). (c) Recognition of THF by the three-way junction site. (d) Recognition of THF by the pseudoknot site.

Figure 8

Structure of the xpt guanine riboswitch colored by the ΔT_m,app of each nucleotide as measured by SHAPE chemical probing. The ΔT_m,app is the difference between the T_m,app measured in the presence of 10 µM hypoxanthine and in the absence of hypoxanthine. Nucleotide positions with a ΔT_m,app< 10 °C are grey, 10 °C < ΔT_m,app< 20 °C are colored yellow, and ΔT_m,app > 20 °C are red. Note that the only nucleotide outside of J2/3 that has a large ligand dependent shift in its T_m,app is U22, whose 2’-hydroxyl group directly participates in a hydrogen bond with the N7 of the purine nucleobase (see Fig. 2c).

Figure 9

Elastic motions of the purine nucleobase as measured by fast fluorescence spectroscopy. (a) Base triples above and below the binding pocket (Fig. 2c) are represented by yellow and blue carbons, respectively. The majority of the population (∼60%) was assigned as that observed in the crystal structure in which the nucleobase is not stacked between the A21–U75 and U22-A52 base pairs. A second population (∼30%) was found to be stacked with A21 and/or A52 (thick black arrow) and a third minor population (∼10%) is stacked with U22 or U75 (thin grey arrow). (b) Side view of the binding pocket with the bases upon which the ligand transiently stack highlighted by asterisks.

Figure 10

Modeling of thermodynamic and kinetic control of riboswitches. (a) Regulatory response curves as a function of adenine concentration as given by equation (2). Each curve represents a specific time required to transcribe the message from the end of the aptamer domain (t=0) until completion of the intrinsic terminator (decision point, t_tx). The curves are calculated using the published values for the association and dissociation rate for the pbuE adenine riboswitch at 35 °C and 1×10⁻¹² M for the RNA concentration (Wickiser et al., 2005a). For these values, t = 6.6 seconds represents 1/koff, and the curve for t = 15 seconds is nearly identical to that calculated for t = ∞. (b) The data from (a) transformed to yield a direct correlation between the T₅₀ and the time the aptamer domain has to equilibrate with the cellular environment (interrogation time). K_D/T₅₀ is the ratio of the measured affinity of the aptamer domain at equilibrium and the calculated concentration of adenine required to elicit a half maximal regulatory response (the fit of the curves in panel (a)). As t_tx increases, this ratio reaches the asymptotic limit of 1, which reflects the aptamer being able to equilibrate during t_tx. It has been suggested that a K_D/T₅₀ value of 0.75 be considered the boundary between the kinetic control (K_D/T₅₀ < 0.75) and thermodynamic control (K_D/T₅₀ > 0.75).

Figure 11

Compounds able to bind to the guanine/adenine classes of the purine riboswitch. (a) Compounds that bind the adenine riboswitch include adenine (1), 2,6-diaminopurine (2), 1,2,4-triazolo-1,2,4-triazole-3,6-diamine (3), 3-bromo-1,2,4-thiadiazol-5-amine (4), 2,4,6-triamino-1,3,5-triazine (5), 2,4,5,6-tetraaminopyrimidine (6). (b) Compounds that bind the guanine riboswitch include guanine (7), hypoxanthine (8), the enol tautomer of xanthine (9), 2-acetoamido-6-hydroxypurine (10), 6-N-hydroxylaminopurine (11), and 2,5,6-triaminopyrimidin-4-one (12). (c) Compounds that bind both riboswitches are 2-aminopurine (13), 6-O-methylguanine (14), and 6-chloroguanine (15).

Figure 12

Crystal structure of azacytosine bound to the engineered binding pocket of the add adenine riboswitch containing a U47C/U51C/U74C mutations (PDB ID 3LA5). Other mutations were introduced at the base of the P2 helix to further increase the RNA’s affinity for azacytosine.