Translational control by diverse RNA folds that recognize the metabolite preQ₁

Typical paradigms of translational regulation utilize proteins, whereas riboswitches are economical, RNA-based elements that govern gene expression without protein partners. Present in all domains of life, riboswitches are commonly located in the 5-untranslated regions of bacterial mRNAs where they establish feedback loops that respond to cellular levels of a specific small-molecule effector. Here we present the novel crystal structure of the recently discovered preQ₁-III (class 3) riboswitch at 2.85 Å resolution. The 101-nucleotide switch features an internal loop pseudoknot (iPK) that coaxially stacks upon flanking helices P2 and P3. PreQ₁ (7-aminomethyl-7-deazaguanine) - the last soluble intermediate in the biosynthesis of the hypermodified tRNA base queuosine (Scheme I) - binds to form a major groove base triple of the form U·preQ₁·C that utilizes highly conserved pyrimidine bases that help define this riboswitch class. This mode of effector binding involves non-canonical Watson-Crick pairing reminiscent of the phylogenetically unrelated preQ₁-II (class 2) riboswitch. Remarkably, both riboswitch classes utilize dual U·A-U base triples that stack against the ligand. The respective nine-nucleotide motifs and their preQ₁ ligands superimpose with an rmsd of 0.4 Å; the class 2 and 3 structures are otherwise unrelated. The class 3 preQ₁ binding pocket also includes contributions from junctions that connect the P2-iPK-P3 coaxial stack with perpendicularly positioned helix P4. Of special interest is the observation that P4 can form a hairpin-loop pseudoknot (hPK) with the ribosome binding site (RBS) of the mRNA, suggesting a mode of ligand-dependent RBS sequestration. Biochemical experiments describing the preQ₁ dependence of hPK formation will be presented, as well as a comparison to known, translational class 1 and 2 preQ₁ riboswitch structures [1-3]. Implications for bacterial translational control will be discussed based on this diverse riboswitch family.