Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Abstract

Prequeuosine (preQ 1 ) riboswitches are RNA regulatory elements located in the 5′ UTR of genes involved in the biosynthesis and transport of preQ 1 , a precursor of the modified base queuosine universally found in four tRNAs. The preQ 1 class II (preQ 1 -II) riboswitch regulates preQ 1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ 1 -II riboswitch bound to preQ 1 . Unlike in the preQ 1 class I (preQ 1 -I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. 13 C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ 1 recognition to block ligand exit and stabilize the binding pocket.Comparison of preQ 1 -I and preQ 1 -II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ 1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ 1 differently, with preQ 1 inserting into different grooves. Our studies show that the preQ 1 -II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.R iboswitches are functional noncoding RNA elements often found at the 5′ end of genes that bind to effector molecules, usually metabolites, to attenuate gene expression (1-8). They generally contain two modular elements, an aptamer that binds directly to a cognate ligand, followed by an expression platform that carries out the regulatory function. Ligand binding induces a conformational rearrangement in the aptamer domain, which is transduced to the expression platform, turning gene expression on or off at the transcription or translation level.Structural studies of diverse riboswitches bound to their cognate ligands have revealed that they have evolved various strategies for high-affinity and specific binding between RNA receptors and ligands. In general, the aptamer domain enfolds the ligand, forming a highly complex 3D conformation using a host of tertiary interactions, including base triples, kink turns, ribose zippers, A-minor motifs, loop-loop interactions, and pseudoknots (9-11). Some riboswitches require divalent cations for ligand binding (9, 12-16), whereas in others divalent cations stabilize the bound state (17-21). Some ligands, including S-adenosyl methionine (22), S-adenosyl homocysteine (22, 23), cyclic di-GMP (24, 25), and prequeuosine (preQ 1 ) (26, 27), have more than one class of riboswitch, with each class adopting a different 3D fold to achieve recognition of the same ligand.Two classes of riboswitches that bind preQ 1 , a precursor t...

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

“…S4). The here observed concentration-independence of class-II riboswitches with respect to binding rates is consistent with the conformational capture model that was deduced from NMR spectroscopic investigations [41]. This model proposed that stem P4 is poised to act as a ‘screw cap’ on preQ 1 recognition to block ligand exit and stabilize the binding pocket.…”

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

“…3A). A notable difference in this comparison is that the hydrogen-bonding pattern between N1 of preQ 1 and the Watson-Crick face of C8 in the preQ 1 -II riboswitch is consistent with bifurcation (12). This mode of imine hydrogen bonding by the ligand is not evident in the preQ 1 -III structure, and appears to be a source of positional differences in the respective structures (Fig.…”

“…Frequent docking of the RBS would sequester the expression platform, leading to ligand-dependent queT gene control by translational attenuation. This paradigm differs from other riboswitches, such as preQ 1 -II and SAM-II, because these molecules integrate RBS sequences directly into their aptamer domains upon ligand binding (8,10,12,16,18). Consequently, RBS docking within these riboswitches is characterized by prolonged high-FRET dwell times in the presence of Mg 2+ and ligand (>2.2 s and ∼3.5 s, respectively) with timescales limited most likely by fluorophore photobleaching (10,16).…”

“…Specifically, preQ 1 binding to the class II riboswitch alters P4 helical dynamics and its proximity to the orthogonally oriented aptamer domain (12,16). Like the preQ 1 -III riboswitch, ITC analysis of preQ 1 -II A-minor adenines verified the importance of these bases in preQ 1 binding, although larger ΔΔG values were observed upon mutagenesis indicative of greater losses in ligand binding (12). Nonetheless, there is notable structural homology between the inclined A-minor adenines of the preQ 1 -II and preQ 1 -III riboswitches ( Fig.…”

“…The preQ 1 -I, preQ 1 -II, S-adenosyl-L-methionine-II (SAM-II), and fluoride riboswitches are representative of this organizational strategy, and their analysis has contributed to a renaissance in our understanding of regulatory pseudoknot structure and dynamics (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). 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.…”