Pseudoknot Preorganization of the PreQ₁ Class I Riboswitch

Abstract

To explore folding and ligand recognition of metabolite-responsive RNAs is of major importance to comprehend gene regulation by riboswitches. Here, we demonstrate, using NMR spectroscopy, that the free aptamer of a preQ(1) class I riboswitch preorganizes into a pseudoknot fold in a temperature- and Mg(2+)-dependent manner. The preformed pseudoknot represents a structure that is close to the ligand-bound state and that likely represents the conformation selected by the ligand. Importantly, a defined base pair mutation within the pseudoknot interaction stipulates whether, in the absence of ligand, dimer formation of the aptamer competes with intramolecular pseudoknot formation. This study pinpoints how RNA preorganization is a crucial determinant for the adaptive recognition process of RNA and ligand.

A neutral pH thermal hydrolysis method for quantification of structured RNAs

“…1). Increases in UV absorbance also have been observed for other riboswitches upon heating (Wickiser et al 2005;Yamauchi et al 2005;Santner et al 2012). The change in UV absorbance is reversible upon cooling, and so corresponds to thermal denaturation of duplex and other types of nucleobase interactions that reduce UV absorbance of the folded RNA.…”

Tuning a riboswitch response through structural extension of a pseudoknot

“…Correspondingly, the preQ 1 -II riboswitch represents a putative target for antibiotic intervention. Although preQ 1 class I (preQ 1 -I) riboswitches have been extensively investigated (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), preQ 1 class II (preQ 1 -II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).…”

GTP Binding Leads to Narrowing of the Conformer Population While Preserving the Structure of the RNA Aptamer: A Site-Specific Time-Resolved Fluorescence Dynamics Study

“…Similar conclusions have come from recent NMR studies of several riboswitch aptamer domains. 33,34 These recent studies point out that several riboswitch aptamer domains preorganize themselves into "bound" conformations in the absence of ligands, 33,34 a scenario leading to strengthening of the conformational selection theory of binding between a ligand and a macromolecule (GTP and aptamer, respectively, in this study). Furthermore, we speculate here by taking a cue from the seminal studies published by Szostak and co-workers on GTP aptamers: a mere 10-fold tighter binding of GTP to an aptamer seems to require ∼10 additional "bits of information" in the structure that in a random sequence space leads to an ∼1000-fold decrease in its abundance.…”

“…Recent NMR studies of aptamer segments of riboswitches, however, have indeed shown preorganization of the aptamer domain into "bound" structure even in the absence of the ligand. 33,34 It is noteworthy that the relative populations of these conformers vary over a wide range from one riboswitch to another. 28−37 The individual lifetime component also has interesting information to offer about the structure of the aptamer and (Table 2) shows that the shortest lifetime (τ 1 ) has a maximal contribution (∼80%) in GTP(4), resulting in the shortest value of τ m compared to those for other locations studied in this work.…”

“…19,22,39,40 Artificially selected RNA aptamers are quite small in size, very similar to the aptamer domain of RNA switches. [28][29][30][31][32][33][34][35][36][37]41,42 In RNA switches, structural changes in the aptamer domain caused by ligand binding led to the switching action. Understanding the intricacies of ligand-induced changes in structure and dynamics in artificial RNA aptamers could, in principle, shed light on the mechanism of the function of RNA switches.…”