Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.

[1]  Maung Nyan Win,et al.  Frameworks for programming biological function through RNA parts and devices. , 2009, Chemistry & biology.

[2]  V. DeRose,et al.  A distance ruler for RNA using EPR and site-directed spin labeling. , 2004, Chemistry & biology.

[3]  T. Logan,et al.  Protein dynamics and monomer-monomer interactions in AntR activation by electron paramagnetic resonance and double electron-electron resonance. , 2007, Biochemistry.

[4]  S. Diekmann,et al.  Distance determination in protein-DNA complexes using fluorescence resonance energy transfer. , 2006, Methods in molecular biology.

[5]  K. Hideg,et al.  Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe , 2006, Nucleic acids research.

[6]  Taekjip Ha,et al.  Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Famulok,et al.  Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy , 2007 .

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

[9]  Eric J. Sorin,et al.  Exploring the helix-coil transition via all-atom equilibrium ensemble simulations. , 2005, Biophysical journal.

[10]  S. Müller,et al.  5-(Benzylmercapto)-1H-tetrazole as activator for 2′-O-TBDMS phosphoramidite building blocks in RNA synthesis , 2002 .

[11]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[12]  B. Epe,et al.  Competition between tetracycline and tRNA at both P and A sites of the ribosome of Escherichia coli , 1987, FEBS letters.

[13]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[14]  Gerrit Groenhof,et al.  GROMACS: Fast, flexible, and free , 2005, J. Comput. Chem..

[15]  E Westhof,et al.  Structural Basis of Ligand Discrimination by Two Related RNA Aptamers Resolved by NMR Spectroscopy , 1996, Science.

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

[17]  S. Sigurdsson,et al.  Nanometer distance measurements on RNA using PELDOR. , 2003, Journal of the American Chemical Society.

[18]  David S. Cafiso,et al.  Identifying conformational changes with site-directed spin labeling , 2000, Nature Structural Biology.

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

[20]  M Yarus,et al.  Diversity of oligonucleotide functions. , 1995, Annual review of biochemistry.

[21]  D. Patel,et al.  Structure, recognition and discrimination in RNA aptamer complexes with cofactors, amino acids, drugs and aminoglycoside antibiotics. , 2000, Journal of biotechnology.

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

[23]  K. Hideg,et al.  Monitoring RNA base structure and dynamics using site-directed spin labeling. , 2003, Biochemistry.

[24]  H. Zimmermann,et al.  DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data , 2006 .

[25]  A. Pardi,et al.  NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer. , 2009, Journal of the American Chemical Society.

[26]  Michael Müller,et al.  Thermodynamic characterization of an engineered tetracycline-binding riboswitch , 2006, Nucleic acids research.

[27]  M. Famulok,et al.  Oligonucleotide aptamers that recognize small molecules. , 1999, Current opinion in structural biology.

[28]  A. Ferré-D’Amaré,et al.  Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch. , 2008, Chemistry & biology.

[29]  J. Szostak,et al.  In vitro selection of functional nucleic acids. , 1999, Annual review of biochemistry.

[30]  Samia Aci-Sèche,et al.  Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling , 2009, Nucleic acids research.

[31]  S. Müller,et al.  RNA double cleavage by a hairpin-derived twin ribozyme. , 2000, Nucleic acids research.

[32]  G. Jeschke,et al.  Dead-time free measurement of dipole-dipole interactions between electron spins. , 2000, Journal of magnetic resonance.

[33]  H. Grubmüller,et al.  Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Wolfgang Hillen,et al.  Gene regulation by tetracyclines. , 2004, Genetic engineering.

[35]  G. Stock,et al.  A PELDOR-based nanometer distance ruler for oligonucleotides. , 2004, Journal of the American Chemical Society.

[36]  O. Delalande,et al.  Double electron-electron resonance (DEER): a convenient method to probe DNA conformational changes. , 2008, Angewandte Chemie.

[37]  Heinz-Jürgen Steinhoff,et al.  A structure-based simulation approach for electron paramagnetic resonance spectra using molecular and stochastic dynamics simulations. , 2006, Biophysical journal.

[38]  G. Jeschke,et al.  Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance. , 2007, Physical chemistry chemical physics : PCCP.

[39]  Barbara Fink,et al.  Molecular analysis of a synthetic tetracycline-binding riboswitch. , 2005, RNA.

[40]  Harald Schwalbe,et al.  Interplay of ‘induced fit’ and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch , 2006, Nucleic acids research.

[41]  H. Steinhoff,et al.  Membrane Protein Structure and Dynamics Studied by Site-Directed Spin-Labeling ESR , 2007 .

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

[43]  C. Spahn,et al.  Throwing a spanner in the works: antibiotics and the translation apparatus , 1996, Journal of Molecular Medicine.

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

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

[46]  B. Robinson,et al.  Site-specific incorporation of nitroxide spin-labels into internal sites of the TAR RNA; structure-dependent dynamics of RNA by EPR spectroscopy. , 2001, Journal of the American Chemical Society.