An energetically beneficial leader–linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was the first example of naturally occurring RNA elements reported to bind small organic molecules cooperatively. Earlier works have shown binding of glycine to the second aptamer allows tertiary interactions to be made between the two aptamers, which facilitates binding of a separate glycine molecule to the first aptamer, leading to glycine-binding cooperativity. Prompted by a distinctive protection pattern in the linker region of a minimal glycine riboswitch construct, we have identified a highly conserved (>90%) leader–linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. In >50% of the glycine riboswitches, the leader–linker interaction forms a kink-turn motif. Characterization of three glycine ribsowitches showed that the leader–linker interaction improved the glycine-binding affinities by 4.5- to 86-fold. In-line probing and native gel assays with two aptamers in trans suggested synergistic action between glycine-binding and interaptamer interaction during global folding of the glycine riboswitch. Mutational analysis showed that there appeared to be no ligand-binding cooperativity in the glycine riboswitch when the leader–linker interaction is present, and the previously measured cooperativity is simply an artifact of a truncated construct missing the leader sequence.