Comparison of a PreQ₁ riboswitch aptamer in metabolite-bound and free states with implications for gene regulation

Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ₁ riboswitch from Thermoanaerobacter tengcongensis in the preQ₁-bound and free states. Although the mode of preQ₁ recognition is similar to that observed for preQ₀, surface plasmon resonance revealed an apparent K _D of 2.1 ± 0.3 nM for preQ₁ but a value of 35.1± 6.1 nM for preQ₀. This difference can be accounted for by interactions between the preQ₁ methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ₁-binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ₁ riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.