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

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.

[1]  Jerry Pelletier,et al.  Inhibition of translation by RNA-small molecule interactions. , 2002, RNA.

[2]  Beatrix Suess,et al.  Screening for engineered neomycin riboswitches that control translation initiation. , 2007, RNA.

[3]  H. Schwalbe,et al.  Structures of RNA switches: insight into molecular recognition and tertiary structure. , 2007, Angewandte Chemie.

[4]  A. J. Shaka,et al.  Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients , 1995 .

[5]  Barbara Fink,et al.  Conditional gene expression by controlling translation with tetracycline-binding aptamers. , 2003, Nucleic acids research.

[6]  C. Hammann,et al.  Comparative thermodynamic analysis of cyclic nucleotide binding to protein kinase A , 2007, Biological chemistry.

[7]  R. Breaker,et al.  The structural and functional diversity of metabolite-binding riboswitches. , 2009, Annual review of biochemistry.

[8]  J H Lakey,et al.  Heat does not come in different colours: entropy-enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions. , 2001, Biophysical chemistry.

[9]  T. Vernet,et al.  A family of yeast expression vectors containing the phage f1 intergenic region. , 1987, Gene.

[10]  Hilla Peretz,et al.  Ju n 20 03 Schrödinger ’ s Cat : The rules of engagement , 2003 .

[11]  D. Patel,et al.  Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer. , 1999, Structure.

[12]  B. Suess,et al.  PELDOR spectroscopy reveals preorganization of the neomycin-responsive riboswitch tertiary structure. , 2010, Journal of the American Chemical Society.

[13]  F. Studier,et al.  Cloning and expression of the gene for bacteriophage T7 RNA polymerase. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[14]  F. Narberhaus,et al.  Microbial thermosensors , 2009, Cellular and Molecular Life Sciences.

[15]  M. Kimmel,et al.  Conflict of interest statement. None declared. , 2010 .

[16]  Beatrix Suess,et al.  Aptamers and riboswitches: perspectives in biotechnology , 2009, Applied Microbiology and Biotechnology.

[17]  C. Wilson,et al.  Inducible regulation of the S. cerevisiae cell cycle mediated by an RNA aptamer-ligand complex. , 2001, Bioorganic & medicinal chemistry.

[18]  S. Brodie,et al.  Promoter-Specific Shifts in Transcription Initiation Conferred by Yeast TFIIB Mutations Are Determined by the Sequence in the Immediate Vicinity of the Start Sites , 2001, Molecular and Cellular Biology.

[19]  B. Suess,et al.  Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch. , 2010, Angewandte Chemie.

[20]  Beatrix Suess,et al.  NMR resonance assignments of an engineered neomycin-sensing riboswitch RNA bound to ribostamycin and tobramycin , 2010, Biomolecular NMR assignments.

[21]  Karl-Dieter Entian,et al.  A fast and efficient translational control system for conditional expression of yeast genes , 2009, Nucleic acids research.

[22]  A. Serganov.  The long and the short of riboswitches. , 2009, Current opinion in structural biology.

[23]  J. Wöhnert,et al.  The structure of the stemloop D subdomain of coxsackievirus B3 cloverleaf RNA and its interaction with the proteinase 3C. , 2004, Structure.

[24]  Joy Sinha,et al.  Reprogramming Bacteria to Seek and Destroy a Herbicide , 2010, Nature chemical biology.

[25]  H. Schwalbe,et al.  NMR Spectroscopy of RNA , 2003, Chembiochem : a European journal of chemical biology.

[26]  Beatrix Suess,et al.  Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast , 2007, Nucleic acids research.

[27]  M. Famulok,et al.  A novel RNA motif for neomycin recognition. , 1995, Chemistry & biology.

[28]  D. S. Pilch,et al.  Thermodynamics of aminoglycoside-rRNA recognition. , 2003, Biopolymers.

[29]  D. Patel,et al.  Adaptive recognition by nucleic acid aptamers. , 2000, Science.

[30]  F. Winston,et al.  A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. , 1987, Gene.

[31]  B. Suess,et al.  Engineered riboswitches: Overview, problems and trends , 2008, RNA biology.

[32]  R. Montange,et al.  Riboswitches: emerging themes in RNA structure and function. , 2008, Annual review of biophysics.

[33]  M. Green,et al.  Controlling gene expression in living cells through small molecule-RNA interactions. , 1998, Science.

[34]  P. Dumas,et al.  Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion , 2007, Nucleic acids research.

[35]  Barbara Fink,et al.  Tetracycline‐aptamer‐mediated translational regulation in yeast , 2003, Molecular microbiology.

[36]  Adelheid Lempradl,et al.  Monovalent Ion Dependence of Neomycin B Binding to an RNA Aptamer Characterized by Spectroscopic Methods , 2007, Chembiochem : a European journal of chemical biology.