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Conformational dynamics of the tetracycline-binding aptamer

By Ute Förster, Julia E. Weigand, Peter Trojanowski, Beatrix Suess and Josef Wachtveitl

Abstract

The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer

Topics: RNA
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:3287181
Provided by: PubMed Central

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Citations

  1. (2009). A fast and efficient translational control system for conditional expression of yeast genes.
  2. (2008). A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli d-3-phosphoglycerate dehydrogenase.
  3. (2001). A tetracycline-binding RNA aptamer.
  4. (2001). Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and conformational capture.
  5. (1991). Determination of the equilibrium association constant between tet repressor and tetracycline at limiting Mg 2+ concentrations: A generally applicable method for effector-dependent high-affinity complexes.
  6. (2009). DynaFit–a Software Package for Enzymology.
  7. (2010). Fast and ultrafast spectroscopic investigation of tetracycline derivatives in organic and aqueous media.
  8. (2003). Fluorescence kinetics of aqueous solutions of tetracycline and its complexes with Mg 2+ and Ca 2+.
  9. (2010). Folding of a transcriptionally acting preQ1 riboswitch.
  10. (2006). Folding of the adenine riboswitch.
  11. (2010). Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch.
  12. (2007). Interplay of ‘induced fit’ and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch.
  13. (2011). Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.
  14. (2007). Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach.
  15. (2007). Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain.
  16. (2005). Molecular analysis of a synthetic tetracycline-binding riboswitch.
  17. (1974). New look at statistical-model identification.
  18. (2009). NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer.
  19. (1991). Organic photochemistry. 93. Photochemical and photophysical studies of tetracycline.
  20. (2009). Photophysics of 1-ethynylpyrene-modified RNA base adenine.
  21. (1970). Photophysics of Aromatic Molecules. Wiley-VCWiley-Interscience,
  22. (1999). Principles of Fluorescence Spectroscopy. 2nd edn.
  23. (1996). Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase.
  24. (2008). RNA dynamics: it is about time.
  25. (2003). Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex.
  26. (2008). Screening for engineered neomycin riboswitches that control translation initiation.
  27. (1992). Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.
  28. (2008). Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch.
  29. (2007). Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast.
  30. (2003). Tetracycline-aptamer-mediated translational regulation in yeast.
  31. (2005). The kinetics of ligand binding by an adenine-sensing riboswitch.
  32. (2005). The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch.
  33. (2006). Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain.
  34. (2006). Thermodynamic characterization of an engineered tetracycline-binding riboswitch.
  35. (2007). Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution.
  36. (1993). Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 2. Quantitative kinetic analysis.
  37. (2010). Ultrafast dynamics show that the theophylline and 3-methylxanthine aptamers employ a conformational capture mechanism for binding their ligands.

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