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Gene regulation by riboswitches

Gene regulation by riboswitches. 1. Regulation of gene expression 2. Principles and examples of RNA-mediated genetic control 3. Riboswitches: - Organization and properties - Transcription termination (FMN riboswitch) - Translation inhibition (thiamine riboswitch)

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Gene regulation by riboswitches

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  1. Gene regulation by riboswitches 1. Regulation of gene expression 2. Principles and examples of RNA-mediated genetic control 3. Riboswitches: - Organization and properties - Transcription termination (FMN riboswitch) - Translation inhibition (thiamine riboswitch) - RNA cleavage (glmS riboswitch) 4. Summary

  2. mRNA Protein DNA Control of gene expression mRNA degradation • protein degradation • export • protein synthesis • protein folding • mRNA synthesis • mRNA folding Transcription Translation

  3. Principles of RNA-mediated genetic control (I) Transcription termination OFF ON Repression Activation

  4. Principles of RNA-mediated genetic control (II) Translation initiation ON OFF Repression Activation

  5. Signals of RNA-mediated genetic control OFF ON Repression Activation Ribosome Protein RNA Temperature Metabolite

  6. Example 1: Ribosome - Transcription attenuation trp genes of E. coli

  7. Example 2: RNA-binding protein ptsGHI genes of B. subtilis antiterminator

  8. Example 3: Antisense RNA rpoS gene of E. coli

  9. Example 4: tRNA glyQS genes of B. subtilis

  10. Example 5: Temperature prfA gene of L. monocytogenes

  11. Example 6: Metabolite (“Riboswitch”) thi operon of B. subtilis

  12. Organization and properties of riboswitches (I) • conserved genetic control elements in the 5’ untranslated region (UTR) of bacterial mRNAs • are regulated by metabolites: coenzymes (B1, B2, B12), amino acids, purin bases • consist of only RNA • estimation: > 2% of all genes are regulated by riboswitches

  13. Organization and properties of riboswitches (II) • structured binding pocket • high affinity: • high specificity Binding domain (Aptamer): • reacts upon ligand binding by the aptamer • comformational change alters gene expression Regulatory Domain: (Expression platform)

  14. Regulation of riboflavin biosynthesis in B. subtilis • RFN element in mRNAs of genes required for biosynthesis of riboflavin and FMN • FMN required for repression of the ribDEAHT operon of B. subtilis • mutations in the RFN element eliminate FMN-mediated repression of the ribDEAHT operon

  15. Spontaneous hydrolysis of RNA

  16. RNA analysis by in-line probing

  17. The RFN element is a riboswitch Winkler et al. (2002), PNAS99, 15908-13; Mironov et al. (2002), Cell111, 747-756 ribD mRNA

  18. The RFN element is a FMN riboswitch Winkler et al. (2002), PNAS99, 15908-13 apparent KD: 3 M 5 nM 300 nM

  19. The FMN riboswitch terminates transcription Winkler et al. (2002), PNAS99, 15908-13

  20. Termination of transcription • RNAP/DNA/RNA complex (TEC) is exceptionally stable • Transcription is processive: RNAP does not come off the DNA until termination pause site 11 bases from hairpin downstream DNA UUUUUUUU • hairpin inactivates and destabilizes TEC • U’s destabilize hybrid

  21. Single-round transcription (synchronized) Phillip et al. (2001), Methods Enzymol 340, 466-85 displaces RNAP from nonspecific binding sites -[32P]UTP, GTP, ATP nascent RNA paused at the first G of template strand

  22. How does the FMN riboswitch really work? Wickiser et al. (2005), Molecular Cell 18, 49-60 Single round transcription assay • apparent dissociation constant KD:  5 nM • conc. to induce 50 % termination:  500 nM Does transcription kinetics operate the FMN riboswitch?

  23. Role of NusA in transcription termination Borukhov et al. (2005), Mol. Microbiol. 55, 1315-1324 • essential transcription elongation factor (conserved among eubacteria and archaea) • stimulates pausing • can induce anti-termination NusA-induced RNAP pausing provides a mechanism for synchronizing transcription and translation

  24. Role of NusA in transcription termination Borukhov et al. (2005), Mol. Microbiol. 55, 1315-1324

  25. NusA reduces the effective FMN concentration Wickiser et al. (2005), Molecular Cell 18, 49-60 Single round transcription assay NusA increases the fraction of terminated transcripts by 2-fold

  26. Identification of transcriptional intermediates Wickiser et al. (2005), Molecular Cell 18, 49-60 Single round transcription assay

  27. Life time of pause sites Wickiser et al. (2005), Molecular Cell 18, 49-60 Single round transcription assay 60 sec 10 sec • NusA increases lifetimes • increasing concentrations of NTPs descreases lifetimes

  28. Dependence of ligand affinity on transcript length Wickiser et al. (2005), Molecular Cell 18, 49-60 PB mimic PA mimic aptamer

  29. Binding of FMN by transcriptional intermediates Wickiser et al. (2005), Molecular Cell 18, 49-60 PB mimic aptamer FMN binding FMN binding (165 ribD and 230 ribD) antiterminator (244 ribD) apparent KD: 10 nM 100 nM -

  30. Binding of FMN by transcriptional intermediates Wickiser et al. (2005), Molecular Cell 18, 49-60 Fluorescence quenching

  31. Binding of FMN by transcriptional intermediates Wickiser et al. (2005), Molecular Cell 18, 49-60 Fluorescence quenching aptamer PA mimic PB mimic

  32. Binding kinetics of FMN to its aptamers Wickiser et al. (2005), Molecular Cell 18, 49-60 Time-resolved fluorescence quenching [M-1s-1] Estimation of times for binding of 50% of ribD mRNA by FMN: (1 M FMN =excess over mRNA, 37 C) 2 s for 200 ribD 34 s for 230 ribD

  33. Life time of pause sites Wickiser et al. (2005), Molecular Cell 18, 49-60 Synchronized transcription assay 60 sec 10 sec • substantial amount of has reached pause sites within 8 s • majority of mRNAs have reached termination site after 60 s Without the pause sites much more than 1 M FMN would be needed to bind the riboswitch with sufficient speed.

  34. Transcriptional intermediates - Conclusions Wickiser et al. (2005), Molecular Cell 18, 49-60 • Aptamer remains receptive to FMN binding until it is disrupted by the formation of the antiterminator helix.= irreversible event of the riboswitch at low FMN concentrations • longer transcripts have lower affinity • aptamers of transcripts paused at PA and PB have affinities better than the T50 values

  35. Life time of the RNA-FMN complex Wickiser et al. (2005), Molecular Cell 18, 49-60 In vitro transcription observed by fluorescence quenching FMN dissociates only very slowly from the ribD mRNA.

  36. Life time of the RNA-FMN complex Wickiser et al. (2005), Molecular Cell 18, 49-60 Dissociation kinetics of the 165 ribD/FMN complex (100 nM, 25 C) koff = 10-3 s-1  k  10 min

  37. Kinetics of antiterminator helix formation Wickiser et al. (2005), Molecular Cell 18, 49-60

  38. FMN and the antiterminator bind competitively to ribD mRNA Wickiser et al. (2005), Molecular Cell 18, 49-60 Dissociation kinetics of the 230 ribD/FMN complex (150 nM, 25 C): antiterminator • 10-fold excess of ATO over FMN reduces ribD/FMN association by half • kon = 103 M-1s-1= 100-fold slower than typical nucleic acid hybridization times • comparison with other intramolecular helix formations: > 3 s for antiterminator formation

  39. 1 s 5.5s 48.5s >5min Kinetic model for the FMN riboswitch Wickiser et al. (2005), Molecular Cell 18, 49-60 1 M FMN, 25 C: • control by kinetic competition: coupling of regulatory reaction to rate of RNA synthesis • FMN binding occurs primarily while RNA polymerase is paused • Caution: - > half of the in vitro transcripts do not function as predicted - low NTP concentration: thermodynamic control

  40. Regulation of thiamine biosynthesis genes in E. coli

  41. The thi box is a riboswitch

  42. The TPP riboswitch inhibits translation initiation

  43. Structure of the thiamine pyrophosphate riboswitch Serganov et al. (2006), Nature441, 1167-1171

  44. Cleavage of RNA by a riboswitch Winkler and Dann (2006), Nat Struct Mol Biol 13, 569-71

  45. Structure of the glmS riboswitch Klein and Ferre-D'Amare (2006), Science313, 1752-6

  46. The glmS riboswitch has pre-formed coenzyme binding pocket Klein and Ferre-D'Amare (2006), Science313, 1752-6

  47. Proposed catalytic mechanism of the glmS ribozyme

  48. Summary • regulatory elements which control a wide set of metabolic pathways in bacteria • consist solely of RNA • Aptamer domain: high affinity and specific binding of metabolites • Expression platform: regulation of gene expression by control of • - transcription termination: kinetically controlled • - translation initiation • - RNA stability

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