Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

Abstract

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.

Molecular sensing by the aptamer domain of the FMN riboswitch: a general model for ligand binding by conformational selection

“…The globally preorganized and bound-like structure of the free FMN riboswitch aptamer domain is reminiscent of observations of other riboswitches, including the lysine riboswitch (16), the SAM-I, -II and -III riboswitches (13,27–29), the preQ 1 riboswitch from T. tengcongensis (12), and even an artificial neomycin-dependent riboswitch (54,55). At physiological concentrations of magnesium (typically 0.5–1.5 mM) but in absence of ligand, these riboswitches adopt interconverting conformations, some of which are close in structure to the ligand-bound state.…”

New trends in the use of aminoglycosides

“…The binding between NEO and the riboswitch aptamer was explained to be the same as the AGs binding to their natural target at the decoding A-site. 80, 81 As the smallest functional artificial riboswitch identified so far, the 27-nt riboswitch has been further investigated via structural and dynamics approaches, NMR, and pulsed electron-electron double resonance (PELDOR) spectroscopy. These studies revealed that in addition to NEO, the riboswitch element also responds to RIB and TOB.…”

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

“…To properly design and optimize synthetic riboswitches, it is extremely valuable to understand how the aptamer interacts with its ligand(s) in the environment in which it will be applied. Neomycin-B binding aptamers have been successfully selected in yeast and applied as riboswitches (Weigand et al 2011), but little is known about the structures they adopt intracellularly and how they interact with their ligands under these conditions.…”