c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry–related pockets

Abstract

The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger c-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongenesis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo two-fold symmetry, with potential for cross-talk between sites mediated by adjacently-aligned base stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by ITC-based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongenesis ydaO riboswitch, like its B. subtilis counterpart, likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an ‘off’ switch.

Structural analysis of a class III preQ 1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

“…By contrast, pseudoknotted aptamers that do not integrate their expression platforms are less common, and this added complexity can encumber efforts to elucidate how ligand-induced conformational changes regulate gene expression. Such riboswitches include the cyclic-di-adenosine monophosphate, SAM-IV, and SAM-I/IV riboswitches (19)(20)(21)(22), as well as the recently identified preQ 1 -III riboswitch (23).The discovery of a third class of preQ 1 riboswitches makes this ligand second only to SAM in terms of the number of riboswitches that respond to this effector (23), underscoring the importance of sensing this molecule within the cell (6). PreQ 1 is the last free intermediate on the queuosine (Q) anabolic pathway (Fig.…”

“…By contrast, pseudoknotted aptamers that do not integrate their expression platforms are less common, and this added complexity can encumber efforts to elucidate how ligand-induced conformational changes regulate gene expression. Such riboswitches include the cyclic-di-adenosine monophosphate, SAM-IV, and SAM-I/IV riboswitches (19)(20)(21)(22), as well as the recently identified preQ 1 -III riboswitch (23).…”

RNA structure through multidimensional chemical mapping

“…Nevertheless, when the crystal structure was released, the M 2 analysis turned out to be accurate: the crystallized RNA did not show electron density for the P4 tetraloop, and the conserved nucleotides in this region formed a non-canonical internal two-way junction instead of a hairpin stem (Gao & Serganov, 2014;Ren & Patel, 2014).…”

“…For most of the recent blind RNA-Puzzle targets, M 2 data were acquired and shared with all modelers during the prediction period, before crystal structures were released after modeling. These targets included two problems (the D. iridis lariat-capping GIR1 ribozyme and the S. thermophilum adenosylcobalamin riboswitch) recently summarized in the RNA-Puzzles Round II paper (Meyer et al 2014;Miao et al 2015;Peselis & Serganov, 2012) and four others for which crystal structures have since been reported (Ren & Patel, 2014;Suslov, 2015;Trausch et al 2014Trausch et al , 2015.…”

“…Visual inspection of M 2 -DMS [not shown; see (Miao et al 2015)] suggested a tertiary contact involving non-canonical pairs between P9 and P2 (gray) that was indeed observed in the subsequently released crystal structure. (c) M 2 data and secondary structures of the ydaO cyclic-di-adenosine riboswitch, RNA-Puzzle 12 (Gao & Serganov, 2014;Ren & Patel, 2014). RNA was probed in presence of 10 µM c-di-AMP.…”