Published online 11 December 2010 Nucleic Acids Research, 2011, Vol. 39, No. 8 3363–3372 doi:10.1093/nar/gkq946 Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity Julia E. Weigand 1 , Sina R. Schmidtke2,3, Tristan J. Will1, Elke Duchardt-Ferner2,3, Christian Hammann 4 , Jens W o¨hnert2,3 and Beatrix Suess1,* 1RNA Biochemistry, 2RNA Structural Biology, 3Center of Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M. and 4Heisenberg research group ribogenetics, Technical University of Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany Received February 4, 2010; Revised September 29, 2010; Accepted September 30, 2010 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 unre- lated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher de- gree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is neces- sary 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 en- gineered riboswitch. INTRODUCTION Regulation of gene expression at the level of RNA fre- quently exploits the conformational ﬂexibility and func- tional versatility of ribonucleic acids. Riboswitches are genetic regulatory elements which consist solely of RNA. They bind speciﬁcally to small molecule metabolites thereby sensing their intracellular concentrations. Metabolite binding induces conformational changes which lead to modulation in gene expression at different levels, such as transcription, translation, splicing, polyadenylation and degradation (1). Riboswitches exploit direct RNA–ligand interaction and fulﬁll both sensory and regulatory function simultaneously rendering auxiliary protein factors unnecessary. This principle is fa- vorable for the development of conditional gene expres- sion systems. Approaches to develop engineered riboswitches make use of in vitro selected, small molecule-binding RNA aptamers (2,3). These aptamers bind their respective ligand with high afﬁnity and speciﬁcity. Usually, they adopt a unique conformation only upon ligand binding with the ligand becoming an integral part of the complex (4). One approach is to insert aptamers into un- translated regions of eukaryotic mRNAs. Only in complex with its ligand, the aptamer is able to inhibit translation initiation (5–9) (Figure 1A) or pre-mRNA splicing (10). Recently we have shown that aptamer-mediated regula- tion is efﬁcient enough to conditionally regulate essential genes in yeast (11). The striking advantage of this aptamer-mediated regulation is that aptamers can be selected in vitro against nearly any ligand of choice (12). In addition, most aptamers are small and thus represent only a minor perturbation when introduced into the host mRNA. During the last years, it became obvious that only a small fraction of in vitro selected aptamers has the poten- tial to function as riboswitches as described above. Therefore we developed a gfp-based screening system which allowed us to search, within an in vitro selected aptamer pool, for those aptamers which confer neomycin-dependent regulation (13). Screening of aptamers with binding speciﬁcity to the drug neomycin (14) resulted in the identiﬁcation of regulating *To whom correspondence should be addressed. Tel: +49 69 798 29785; Fax: +49 69 798 29323; Email: suess@bio.uni-frankfurt.de The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.