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RNA-metabolite interactions (riboswitches)

RNA-metabolite interactions (riboswitches). RNA aptamers. • RNA aptamers are structures that bind specifically to target ligands • Many aptamers have been generated in the laboratory for a wide range of target molecules ( e.g. theophylline aptamer)

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RNA-metabolite interactions (riboswitches)

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  1. RNA-metabolite interactions (riboswitches)

  2. RNA aptamers • RNA aptamers are structuresthat bind specifically to targetligands • Many aptamers have beengenerated in the laboratoryfor a wide range of targetmolecules (e.g. theophyllineaptamer) • Structural studies of aptamer-ligand complexes have provided a wealth of information regarding RNA structure and ligand interaction • Most aptamers exhibit conformational changes upon binding to ligand (induced fit binding)

  3. Natural aptamers are the basis of riboswitches • Natural aptamers reside in the non-coding segments of messenger RNAs (mainly prokaryotic) • Interact directly (without the need for protein) with metabolites to control gene expression • Typically provide a feedback mechanism for controlling the expression of metabolic genes metabolic product of a pathway inhibits expression of proteins required to produce the metabolite • Most riboswitches are comprised of the aptamer and an ‘expression platform’ (some way of altering gene expression)

  4. Conserved RNA sequence involved in riboflavin synthesis Flavin mononucleotide RFN aptamer Gelfand, M.S., Trends in Genetics (1999) 15: 439-442.

  5. Genetic Control Mechanisms Tom Cech, Nature (2004) 428: 263-264.

  6. Genetic Control Mechanisms - RIBOSWITCHES Tom Cech, Nature (2004) 428: 263-264.

  7. Natural aptamers are the basis of riboswitches

  8. Riboswitches • RNA domains that modulate gene expression in response to metabolite binding Pre-Queuosine-I 2’-deoxyguanosine Winkler & Breaker (2005) Annu Rev Microbiol59: 487-517 Roth, et al. (2007) Nat Struct Mol Biol.14: 308-317 Kim et al. (2007) Proc. Natl. Acad. Sci.104: 16092-16097

  9. Known riboswitch aptamers

  10. Known riboswitch aptamers

  11. Known riboswitch aptamersGlycine riboswitch -use of cooperativity

  12. Known riboswitch aptamersGlucosamine-6-phosphate riboswitch -riboswitch is a ribozyme Tom Cech, Nature (2004) 428: 263-264.

  13. Riboswitches: RNA-mediated genetic control Mandal et al. (2003) Cell113: 577-586 ~4% Winkler & Breaker (2005) Annu. Rev. Microbiol.59: 487-517

  14. Conformation change drives riboswitch function • Conformation changes upon metabolite-binding alter RNA structure and affect gene expression • Three known mechanism of action: Transcription termination/antitermination Translation control RNA processing

  15. Riboswitch mechanisms • Transcription termination and anti-termination • Translation initiation: RBS accessibility • RNA processing: Splicing or degradation Winkler & Breaker (2005) Annu Rev Microbiol 59:487-517.

  16. Riboswitch mechanisms

  17. Barrick & Breaker (2006) Scientific American296: 50-57

  18. TPP Riboswitch • Recognizes ligand phosphate through metal ion-mediated backbone and nucleobase contacts • Riboswitch regulation of gene expression is perturbed by the antibiotic PTPP • Riboswitch agonists or antagonists: novel antibiotics? Sudarsan et al. (2005) Chem Biol12:1325-1335 Serganov et al. (2006) Nature441:1167-1171 Thore et al. (2006) Science312:1208-1211

  19. Thiamine pyrophosphate (TPP) riboswitch

  20. TPP-dependent modulation of spontaneous RNA cleavage In-line attack technique

  21. In-line attack technique

  22. RNA is a high-affinity metabolite receptor Apparent KD based on half maximal modulation of RNA

  23. Equilibrium dialysis technique 3H-thiamine 3H-thiamine

  24. High sensitivity and selectivity of mRNA for metabolite binding Equilibrium dialysis M3 = non-TPP binding mutant

  25. Mutational analysis of the structure & function of thiM riboswitch -gal reporter gene + riboswitch - control of gene expression?

  26. Proposed mechanism for TPP-dependent deactivation of thiM translation

  27. Additional Riboswitch Info

  28. Tandem riboswitches exhibit complex gene control

  29. Diverse tandem riboswitch configurations & functions

  30. Orphan riboswitches

  31. Crystal structure - guanine riboswitch

  32. Crystal structure - guanine riboswitch Guanine would have an amino group here.

  33. Crystal structure - TPP riboswitch

  34. Crystal structure - SAM riboswitch

  35. Crystal structure - GlcN6P riboswitch

  36. Crystal structure – Lysine riboswitch

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