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.

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ₁ riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ₁ aptamer domain in complex with its precursor metabolite preQ₀. The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated.

PreQ₁ riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ₁ during the biosynthesis of the essential tRNA base queuosine. We report what is to our knowledge the first preQ₁-II riboswitch structure at 2.3-Å resolution, which uses a previously uncharacterized fold to achieve effector recognition at the confluence of a three-way helical junction flanking a pseudoknotted ribosome-binding site. The results account for translational control mediated by the preQ₁-II riboswitch class and expand the known repertoire of ligand-binding modes used by regulatory RNAs.

Significance: Riboswitches are RNA molecules found mostly in bacteria that control genes by sensing cellular levels of metabolites, such as the simple organic compound preQ ₁ . The diversity of riboswitches and their potential as novel antibiotic targets continue to elicit interest in these regulatory sequences. Here we present the crystal structure of a newly discovered bacterial preQ ₁ -III riboswitch that senses preQ ₁ using an unusual, two-part architecture. A complementary analysis of flexibility and dynamics showed that recognition of preQ ₁ induces riboswitch compaction, while concomitantly enhancing formation of a distant double-helix possessing a regulatory signal that zips and unzips rapidly, producing gene “off” and “on” states. These observations expand our knowledge of riboswitch construction and suggest a broader role for dynamics than previously recognized.

Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-Å resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site “flap” whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.