Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

Abstract

The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

“…S5). This result is consistent with the findings of a previous study that analyzed the U69A mutation and found the resulting construct to be inactive for glycine binding (46). The lack of observed binding by D,G,A variants, in combination with our bioinformatic analysis, indicates that these RNAs have likely undergone a ligand specificity switch.…”

RNA quaternary structure and global symmetry

“…SAXS experiments indicated that the K-turn-containing riboswitch is more compact than the K-turn-lacking riboswitch even in the absence of glycine, consistent with partial folding of the riboswitch [65]. Recently, mutational analysis of each glycine-binding site has suggested that, although independent and not cooperative, the two ligand sites communicate via pseudo-quaternary contacts α, β, and γ [56] (Fig. 4).…”

“…4C). Despite the relatively modest conservation at these sites, they covary to preserve pairing [38], and disruption of pairing greatly affects riboswitch dimerization [56]. …”

“…The early report [30] that the glycine riboswitch binds its ligands cooperatively appears to be an artifact resulting from analysis of truncated riboswitch constructs lacking a functionally important K-turn motif [38, 39, 56, 65-68]. This K-turn is formed by a long-distance interaction between the 5′ end of the RNA and the linker region connecting the two aptamer domains [67, 68].…”

Modular arrangement of regulatory RNA elements

“…1A). Both aptamers can bind one molecule of glycine in the bulge of helix P3 6 . Cooperative glycine binding leads to dimerization of both aptamers, in which stem P1 of the first aptamer provides the scaffold for the dimer interface.…”