Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch

Eichhorn, Catherine D.; Feng, Jun; Suddala, Krishna C.; Walter, Nils G.; Brooks, Charles L.; Al-Hashimi, Hashim M.

The class I prequeuosine riboswitch (queC), typically found in firmicute bacterial species, is commonly located in the 5 0 -untranslated region (UTR) of the queCDEF operon, which expresses proteins directly involved in the queuosine biosynthetic pathway (25) . The aptamer binds preQ 1 , an intermediate in queuosine synthesis, with high affinity to attenuate protein expression at either the transcription or translation level (25) . This class has the smallest minimal aptamer domain (34 nucleotides, nt) discovered to date, consisting of a small hairpin followed by a 12 nt ssRNA tail ( Figure 1A ). Upon ligand recognition, the highly conserved adenine-rich tail condenses into a pseudoknot, forming a host of interactions to both the hairpin and ligand, including A-minor 'kissing' interactions between the ssRNA polyadenine tract and the minor groove (26) (27) (28) (29) (30) . The activity of transcriptionregulating riboswitches, such as the Bacillus subtilis queC riboswitch, has been shown to depend on the kinetics of ligand binding as well as the rate of transcription (8) . Notably, the very small size of the queC riboswitch leaves very little time, in comparison to other switches, for ligand binding to take place prior to formation of the anti-terminator helix which, when formed, prevents terminator helix formation, thereby allowing gene expression to continue. For example, the B. subtilis FMN riboswitch, which is highly dependent upon the rate of polymerase and contains sites that locally pause polymerase to lengthen the ligand-binding window, has $70 nt between the minimal aptamer sequence and complete formation of the anti-terminator helix (8) . In comparison, the ligand-binding window for the queC riboswitch is $20 nt (26, 27) . How efficient ligand binding is achieved is unclear given that the ssRNA tail is thought to be highly disordered, and therefore capable of sampling a wide range of competing conformations.